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National Academies of Sciences, Engineering, and Medicine; National Academy of Medicine; National Academy of Sciences; Committee on Human Gene Editing: Scientific, Medical, and Ethical Considerations. Human Genome Editing: Science, Ethics, and Governance. Washington (DC): National Academies Press (US); 2017 Feb 14.

Human Genome Editing: Science, Ethics, and Governance.

• Hardcopy Version at National Academies Press

3 Basic Research Using Genome Editing

The recent remarkable advances in methods for editing the DNA of genes and genomes have engendered much excitement and activity and had a major impact on many areas of both basic and applied research. It has been known for 60 years that all life on Earth is encoded in the sequence of DNA, which is inherited in each succeeding generation, but accelerating advances have greatly enhanced understanding of and the ability to manipulate DNA.

This chapter reviews the various types of and purposes for basic laboratory research involving human genome editing. It begins by describing the basic tools of genome editing and the rapid advances in genome-editing technology. The chapter then details how genome editing can be used in basic laboratory research aimed at advancing understanding of human cells and tissues; of human stem cells, diseases, and regenerative medicine; and of mammalian reproduction and development. Ethical and regulatory issues entailed in this research are then summarized. Throughout the chapter, key terms and concepts germane to basic research involving genome editing are defined; Box 3-1 defines the most foundational of these terms.

Foundational Terms.

• THE BASIC TOOLS OF GENOME EDITING

All living organisms, from bacteria to plants to humans, use similar mechanisms to encode and express genes, although the sizes of their genomes and their numbers of genes differ greatly. Hence, understanding of any form of life is immensely informative with respect to understanding all other forms, and provides insights and applications that obtain across species—a fact that has been particularly invaluable in the development of methods for editing genes and genomes.

The earliest studies in molecular biology were on bacteria and their viruses. Their relative simplicity and ease of analysis were key in establishing the basis of the genetic code and the expression of genes. Parallel research on more complex organisms built on the advances in these studies of bacteria, and by the mid-1960s, it was clear that bacteria, plants, and animals shared many fundamental molecular mechanisms. Key discoveries in bacteria uncovered some of their mechanisms for protection against viruses, including so-called restriction endonucleases, proteins bacteria use to cleave the DNA of infecting viruses and “restrict” their growth. This discovery allowed scientists to cut DNA in predictable and reproducible ways and to reassemble the cut pieces into recombinant DNA.

By the mid-1970s, it was evident that recombinant DNA offered a powerful means of combining DNA in productive ways, with promising applications in biotechnology. However, this potential also raised questions about whether the application of these novel methods might entail some risk. In light of those concerns, a group of scientists and others convened a meeting at Asilomar in 1975 to consider what precautions might be needed to oversee this new technology and established a set of guidelines to regulate the containment and conduct of the research. The descendants of those guidelines still regulate recombinant DNA research to this day, some of them incorporated into official regulatory systems. In practice, the most extreme concerns did not eventuate. Today, the use of recombinant DNA methods is widespread worldwide and has yielded enormous benefits to humankind in terms of scientific understanding and medical advances, including many valuable drugs and treatments, and the biotechnology industry is now a thriving part of the world economy.

Among methods developed through the use of recombinant DNA technology is the ability to introduce DNA into cells where it can be expressed—a so-called transgene. This method is widely used in fundamental laboratory research (see Appendix A for more detail). When such exogenous DNA is introduced into a cell, it can insert into the DNA of the cell's genome largely at random and, depending on how and where it is inserted, can be expressed as RNA and protein, although this overall process is not very efficient. A key advance was the development of techniques for generating molecular tools that could be used to cut the DNA of genes and genomes in specific places to allow targeted alterations in the DNA sequence. It was found that double-strand breaks (DSBs) could be deliberately generated by nucleases that cut DNA at defined sites ( homing nucleases , sometimes also called meganucleases , originally discovered in yeast) ( Choulika et al., 1995 ; Roux et al., 1994a , b ). In the succeeding 20 years, based on these groundbreaking discoveries, several additional types of nucleases that can be targeted to specific sites were developed and adapted for use in targeted DNA cleavage ( Carroll, 2014 ).

Such double-strand breaks also occur naturally during DNA replication or through radiation or chemical damage, and cells have evolved mechanisms for repairing them by rejoining the ends (a process known as nonhomologous end joining [NHEJ ]). However, this rejoining often is not perfect, and small insertions and deletions can be introduced during the repair. Such insertions and deletions ( indels ) can disrupt the sequence of the DNA and often inactivate the gene that was cut. This targeted cleavage and inaccurate repair through NHEJ provide a means of inactivating genes or gene-regulatory elements. Although the resulting indels are usually one or a few nucleotides long, in some cases they can consist of thousands of base pairs. Genome editing through NHEJ can also be harnessed to create defined chromosomal deletions or chromosomal translocations by simultaneously creating two double-strand breaks at different sites, followed by rejoining at those two sites. These sites can be either on the same chromosome (producing a deletion) or on different chromosomes (producing a translocation).

More precise editing can be achieved if, during the breakage-repair process in the cell, an extra piece of DNA is provided that shares sequence (i.e., is homologous) with the cleaved DNA. Such homologous repair also is used by normal cellular repair mechanisms. These mechanisms can be exploited to make precise changes. If homologous DNA slightly different in sequence from the cleaved sequence is introduced into the cell, that difference can be inserted into the sequence of the gene or genome, a process termed homology-directed repair (HDR) . HDR can also be used to insert a novel sequence (e.g., one or more genes) of variable length at a precise genomic location. In contrast to NHEJ, HDR-mediated genome editing allows scientists to predict both where the edit will occur and the size and sequence of the resulting change. Thus, HDR-mediated editing is very much like editing a document because precise changes in the characters can be made.

Two types of targeted nucleases that have been widely developed for use in editing genes and genomes are zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) . Both rely on proteins whose normal function is to bind to specific relatively short DNA sequences. Zinc fingers are segments of proteins used by multicellular organisms to control the expression of their genes by binding to DNA (they also typically bind zinc as part of their structure; hence their name). They can be engineered by molecular biologists to recognize different short DNA sequences and can be joined to nucleases that cleave DNA. Thus, the zinc fingers target specific sequences in genes and genomes, and the attached nucleases cleave the DNA to generate a double-strand break by cleaving both strands of the DNA. ZFNs have been developed for gene editing and are in clinical trials—for example, in attempts to confer resistance to the HIV virus in AIDS patients ( Tebas et al., 2014 ). TALENs work similarly to ZFNs, also using DNA recognition proteins (transcription activator-like effectors or TALEs) originally identified in bacteria that infect plants. The DNA recognition sequences of TALE proteins are made of repeating units, each of which recognizes a single base pair in the DNA. TALEs are simpler and easier to engineer than are zinc fingers and can similarly be joined to DNA-cleaving nucleases to yield TALENs. The preclinical application of TALENs to engineer lymphocytes for the treatment of acute lymphoblastic leukemia was recently reported ( Poirot et al., 2015 ).

Thus, these tools are already well-established approaches to the use of genome editing for applications in gene therapy, and many of the associated safety and regulatory issues have already been addressed (see Chapter 4 ). However, the protein engineering required to design site-specific versions of TALENs and, even more so, of ZFNs, remains technically challenging, time-consuming, and expensive.

The past 5 years have seen the development of a completely novel system, known as CRISPR/Cas9 (CRISPR stands for clustered regularly interspaced short palindromic repeats) ( Doudna and Charpentier, 2014 ; Hsu et al., 2014 ). Short RNA sequences modeled on the CRISPR system, when paired with Cas9 (CRISPR associated protein 9, an RNA-targeted nuclease), or alternatively with other similar nucleases, can readily be programmed to edit specific segments of DNA. The CRISPR/Cas9 system is simpler, faster, and cheaper relative to earlier methods and can be highly efficient. CRISPR/Cas9, like TALEs, was originally discovered in bacteria, where it functions as part of an immunity system to protect bacteria from invading viruses ( Barrangou and Dudley, 2016 ; Doudna and Charpentier, 2014 ). The key distinguishing feature of CRISPR/Cas9 is that it uses RNA sequences instead of protein segments to recognize specific sequences in the DNA by complementary base pairing.

As first reengineered in 2012 ( Jinek et al., 2012 ), the bacterial nuclease Cas9 binds a single RNA sequence known as a guide RNA tailored to recognize any sequence of choice. This two-component system can bind to the chosen site in DNA via the guide RNA and cleave the DNA using the Cas9 nuclease. Since it is simple to synthesize RNA of any desired sequence, generation of CRISPR/Cas9 targeting nucleases is straightforward—the system is readily programmed to target any sequence in any genome. Programs exist for choosing suitable guide RNAs, and while not all guides work equally well, testing a number of guides to find effective ones is not difficult or expensive. This ease of design, together with the remarkable specificity and efficiency of CRISPR/Cas9 has revolutionized the field of genome editing and has major implications for advances in fundamental research, as well as in such applications as biotechnology, agriculture, insect control, and gene therapy.

Figure 3-1 provides a summary of the ZFN, TALEN, and CRISPR methods of genome editing. As mentioned, these genome-editing methods are being widely applied across a broad range of biological sciences, from fundamental laboratory research on cells and laboratory animals; to applications in agriculture involving improvements in crop plants and farm animals; to applications in human health, both at the research level and, increasingly, in clinical applications. Agricultural applications have been addressed in other studies by the U.S. National Academies of Sciences, Engineering, and Medicine (see Chapter 1 ) and potential clinical applications are the subject of subsequent chapters of this report. The focus in this chapter is on basic laboratory research using genome editing.

Methods of genome editing. Top: Zinc finger nucleases (ZFNs): The colored modules represent the Zn fingers, each engineered to recognize three adjacent base pairs in the DNA; these modules are coupled to a dimer of the FokI nuclease that makes a double-stranded (more...)

This research addresses fundamental questions concerning the use and optimization of genome-editing methods both in cultured cells and in experimental multicellular organisms (e.g., mice, flies, plants). Such basic discovery research is essential for improving any future applications of genome editing. Applications of genome editing in laboratory research also have added powerful new tools that are contributing greatly to understanding of basic cellular functions, metabolic processes, immunity and resistance to pathological infections, and diseases such as cancer and cardiovascular disease. These laboratory studies are overseen by standard laboratory safety mechanisms. In addition to these applications, this chapter reviews the potential for using similar approaches in basic research on human germline cells, not for the purposes of procreation but solely for laboratory research. This work will provide valuable insights into the processes of early human development and reproductive success, and could lead to clinical benefits, directly as a result of work with human embryos and germline cells or through improvements in the derivation and maintenance of stem cells in vitro.

• RAPID ADVANCES IN GENOME-EDITING TECHNOLOGY

The development of CRISPR/Cas9 has revolutionized the science of gene and genome editing, and the basic science is advancing extremely rapidly, with additional CRISPR-based systems being developed and deployed for multiple different purposes. Different species of bacteria use somewhat different CRISPR systems, and although the CRISPR/Cas9 system is currently the most widely used because of its simplicity, alternative systems being developed will provide increased flexibility in methodology ( Wright et al., 2016 ; Zetsche et al., 2015 ).

Among the issues that need to be addressed going forward are the specificity and efficiency of the DNA cleavage mediated by CRISPR-guided nucleases. While the roughly 20-base sequence recognized by the guide RNA provides a great deal of specificity (an exact match should occur by chance in approximately 1 × 10 12 base pairs—1 in a trillion—the equivalent of several hundred mammalian genomes), there is some small potential for so-called off-target events , in which the nucleases make cuts in unintended places, especially if the guide RNA binds to DNA sequences that are slightly different from the intended target. Some early experiments suggested that off-target events might occur at a significant rate, but as the methods have been improved and as their application has increasingly been in normal cells rather than cultured cell lines, the frequency of off-target cleavages appears to be very low. Advances have been achieved in the specificity of Cas9 cleavage ( Kleinstiver et al., 2016 ; Slaymaker et al., 2016 ), and methods have been developed for monitoring the frequency of off-target cleavage. (See Appendix A for more detail.)

Another significant advance has occurred in the development of methods for modifying the CRISPR/Cas9 system so that DNA cleavage is avoided. For example, the nuclease function of Cas9 can be inactivated so that a complex of guide RNA and such a “dead” Cas9 (dCas9) will target a specific site via the guide RNA but will not cleave the DNA ( Qi et al., 2013 ). By coupling other proteins with different activities to the dCas9, however, different sorts of modifications can be made to the DNA or its associated proteins. Thus, it is possible to design variants of CRISPR/Cas9, ZFN, or TALE that will turn on or turn off adjacent genes, make single-base changes, or modify the chromatin proteins that associate with DNA in chromosomes and thus modify the epigenetic regulation of genes ( Ding et al., 2016 ; Gaj et al., 2016 ; Konerman et al., 2015 ; Sander and Joung, 2014 ). All of these noncleaving variants fail to cleave DNA, thus reducing the potential for deleterious off-target events, and many other modifications are being introduced to enhance specificity and reduce off-target events (see Appendix A for further detail). Most recently, CRISPR/C2c2, a programmable RNA-guided, RNA-cleaving nuclease, has been described ( Abudayyeh et al., 2016 ; East-Seletsky et al., 2016 ) that could be used to knock down specific RNA copies of genes without affecting the gene itself. This development raises the future possibility of nonheritable or reversible editing.

As can be seen from this brief survey, the rapidly developing versatility of these RNA-guided genome-editing systems is opening up numerous means of manipulating the expression and function of genes. A recent report of methods for inducibly knocking down or knocking out genes in a multiplex fashion in many cell types, including human pluripotent stem cells, as well as in mice ( Bertero et al., 2016 ) further expands the potential of these methods. These and other advances have rapidly rendered these methods basic tools of molecular biology worldwide, adding to the existing toolkit assembled over the past 40 years. These methods are now being applied to study with unprecedented ease the functions of genes in cells and in experimental animals, such as yeast, fish, mice, and many others, to enhance understanding of life. They also are being used to investigate the derivation and differentiation of stem cells, providing fundamental insights relevant to regenerative medicine, and to develop culture models of human disease both to advance understanding of disease processes and to enable testing of drugs on human cells ex vivo.

• BASIC LABORATORY RESEARCH TO ADVANCE UNDERSTANDING OF HUMAN CELLS AND TISSUES

Basic biomedical research aimed at discovering more about the mechanisms and capabilities of genome editing offers significant opportunities to advance human medicine. Genome-editing research conducted on human cells, tissues, embryos, and gametes in the laboratory offers important avenues for learning more about human gene functions, genomic rearrangements, DNA-repair mechanisms, early human development, the links between genes and disease, and the progression of cancer and other diseases that have a strong genetic basis. Manipulation of genes and gene expression by genome editing allows one to understand the functions of genes in the behavior of human cells, including why they malfunction in disease. For example, editing of cultured human cells to model the changes that arise in cancer or in genetically inherited diseases provides culture models of those diseases with which to understand the molecular basis of the resulting defects. Such laboratory studies also allow the development of means of combating those defects, such as the testing of potential drugs in cell culture. All of those approaches are much easier now than they were just a few years ago.

Certain cells derived from an early embryo, after fertilization but prior to the developmental stage at which it would implant in a woman's uterus, are referred to as embryonic stem (ES) cells. These ES cells have scientific advantages because they can reproduce in cell culture and have the potential to form all the different body cell types while lacking the potential themselves to develop into a fetus. It is now also possible to create stem cells by manipulating adult somatic cells to convert them to a state in which they, too, have the ability to form multiple cell types, reducing the need to take stem cells from an early embryo. These are referred to as induced pluripotent stem (iPS) cells. Such pluripotent stem cells can be cultured in vitro and induced to develop into many different cell types, such as neurons, muscle or skin cells, and many others. Advances over the past several decades in understanding stem cells and how they can be used form the foundation for the field of regenerative medicine, which seeks to repair or replace damaged cells within human tissues or to generate new tissues after disease or injury. Although these are increasingly areas of clinical practice, and the application of genetically altered cells in humans is not covered in this chapter (see Chapter 4 ), there are nevertheless a number of important reasons why scientists aim to undertake basic investigations in human and animal stem cells in the laboratory.

Genome-editing methods have been extremely useful in generating a variety of genetic modifications in human ES and iPS cells. Before the advent of efficient genome-editing tools, these cells had proven resistant to genetic modification with the standard tools of homologous recombination that had been used effectively in mouse ES cells. Using those tools in human cells resulted in very low frequencies of targeted recombination. Improvements in efficiency resulting from the use of CRISPR/Cas9 have enabled rapid generation of tagged reporter cell lines, making it possible to follow differentiation pathways, look for interacting proteins, sort appropriate cell types, and investigate the functions of individual genes and pathways in cells, among many other applications ( Hockemeyer and Jaenisch, 2016 ). For example, the ability to make precisely targeted mutations or corrections in specific genes has made possible the generation of human ES lines with different specific disease alleles on the same genetic background ( Halevy et al., 2016 ) for use in research on the consequences of such disease genes. Conversely, genome editing also allows the targeted correction of disease mutations in patient-specific iPS cell lines to generate genetically matched control lines. Such modified stem cell lines are used primarily to conduct experimental and preclinical studies, to investigate specific disease processes, and to test drugs that could be used to treat such diseases. In the future, such edited stem cell lines could be used for various forms of somatic cell–based therapies (see Chapter 4 ).

• BASIC LABORATORY RESEARCH TO ADVANCE UNDERSTANDING OF MAMMALIAN REPRODUCTION AND DEVELOPMENT

Germline cells are cells with the capacity to be involved in forming a new individual and to have their genetic material passed on to a new generation. They include precursor cells that form eggs and sperm, as well as the eggs and sperm cells themselves. When fertilization occurs to create an embryo, the earliest stages of this embryo, referred to as the zygote (fertilized egg) and blastocyst, have the potential to divide and form all the cells that will make up the future individual, including somatic (body) cells and new germ cells. As the embryo continues to develop, its cells differentiate into specific cell types that become increasingly restricted in their functions (e.g., to form specialized cells such as those in the nervous system, skin, or gut).

During reproduction and development, genetic changes made directly in gametes (egg and sperm), in egg or sperm precursor cells, or in very early embryos would be propagated throughout the future cells of an organism and may therefore be heritable by subsequent generations. As emphasized above, this chapter focuses exclusively on the use of genome-editing technologies in the laboratory, and not on clinical applications in humans or in embryos for the purposes of implantation to initiate pregnancy. Nevertheless, it is important to understand which cell types are involved in human development and their functions, because this information informs researchers' decisions about how to study particular scientific questions and informs ethical, regulatory, and social discussions around when and why it may be useful to use human cells, including embryos, in basic laboratory research.

Genome Editing of Germline Stem Cells and Progenitor Cells

It is already possible in mice to genetically modify the genome in a fertilized egg (the zygote), in individual cells of the early embryo, in pluripotent ES cells, or in spermatogonial stem cells, just as in somatic cells. In all these cases, the effects of the genetic modifications can be studied directly in the embryo or in cells in culture. There are a number of ways to undertake these genetic manipulations and a number of cell types in which they can be conducted. The cell types below are all considered part of the germline or have the capacity to contribute to the germline:

• embryonic stem cells derived from normal early embryos (blastocyst stages)
• cells from early embryos produced after somatic cell nuclear transfer (SCNT) 1
• iPS cells obtained by reprogramming somatic cells into an ES celllike state

In mice, these cell types can all be manipulated experimentally through genome editing. Stem cells of the types listed above can contribute to the germline in vivo after they are introduced into mouse embryos at the morula or blastocyst stage. This process generally creates an embryo that is a chimera, in which some cells are derived from the stem cells introduced into the embryo, and some are formed from the initial embryonic cells. Mouse or rat spermatogonial stem cells can be cultured and their genomes edited, and the cells can then be introduced into recipient mouse or rat testes, where they can give rise to sperm able to fertilize oocytes, at least in vitro (see Appendix A and Chapman et al., 2015 ). In all of these cases, when the resultant embryos are transferred back into the uterus to complete pregnancy, it is possible to establish lines of mice carrying the genetic alterations. These approaches provide unprecedented opportunities to explore the functions of all the genes in the genome and to develop rodent models of human diseases. Proof-of-principle experiments also have been reported in which disease-related genetic mutations have been corrected in mouse zygotes ( Long et al., 2014 ; Wu et al., 2013 ), embryonic stem cells, or spermatogonial stem cells ( Wu et al., 2015 ) and then transmitted though the germline to produce genetically corrected mice.

The application of genome-editing technologies to the equivalent human cell types holds considerable potential value for fundamental research without any intent to use such manipulated cells for human reproductive purposes. Improved knowledge of how an early human embryo develops also is valuable in its own right, and because such knowledge can help answer questions about humans' own early development, as well as facilitate understanding and potential prevention or treatment of a wide range of clinical problems. A number of these applications are described below.

Improvements in Assisted Reproductive Technology

The success of human reproductive technologies and preimplantation genetic diagnosis (PGD) of inherited diseases has been, and continues to be, dependent on in vitro fertilization (IVF) and on culturing of human embryos from the zygote to the blastocyst stage. However, tools for ensuring that an individual embryo in culture is normal and capable of completing pregnancy remain limited. Most embryo research has been conducted on mouse embryos, which are similar to human embryos in certain respects but significantly different in others (see Box 3-2 ). Even the conditions in which human embryos are kept in culture are based largely on those established for mouse embryos. High rates of aneuploidy 2 are found in cultured human embryos relative to other species. This aneuploidy is often mosaic—that is, it varies among cells in the embryo ( Taylor et al., 2014 )—but how it arises and how it relates to in vitro culture conditions are not well understood. There is also concern that epigenetic 3 abnormalities might occur in human embryos in vitro ( Lazaraviciute et al., 2014 ), which might compromise development or health, even later in life. Research on early-stage human embryos in culture should enable scientists to better understand the cellular and molecular pathways that control early human embryo development and the conditions under which human embryos in culture can develop successfully. This knowledge could in turn help improve IVF outcomes.

Differences Between Mouse and Human Development.

All of the differences between humans and mice discussed above mean that it is not possible to accurately infer developmental events in human embryos from studying mice. This limitation has practical consequences for the development of improved IVF technologies, as well as for the ability to derive the best pluripotent or other stem cells for modeling of human disease and for future regenerative therapies. Thus, there is considerable interest in experimental investigation of preimplantation human development in culture, in jurisdictions where such research on human embryos is permitted. The goals of this work are to understand the fundamental events of fertilization, activation of the embryonic genome, cell lineage development, epigenetic events such as X-inactivation, and others, and how these events compare and contrast with what is understood from studying mice.

Similar research also could provide insights into the reasons for the high rates of early pregnancy loss in natural human pregnancies (10 to 45 percent, depending on the age of the mother), as well as the causes of infertility. Better understanding of sperm development would be crucial in addressing issues of male infertility. Pluripotent stem cells arise from the early embryo, and these cells can generate ES cells in culture. Better understanding of human embryonic development would provide insights into the origins and regulation of pluripotency and how to translate that knowledge into improved stem cells for regenerative medicine. The potential benefits of such research are not limited to embryonic stem cells. Cell types that give rise to the yolk sac and the placenta also are determined in the early embryo prior to implantation. The yolk sac and placenta establish the crucial links with the mother during pregnancy and provide nutrients and other factors that enable the embryo to survive. Defects in these tissues can compromise a pregnancy, leading to miscarriage, premature birth, or postnatal abnormalities. Better understanding of how the yolk sac and placenta originate would help in improving techniques for overcoming infertility and preventing early miscarriage, as well as understanding and preventing congenital malformations. These extraembryonic cell types also provide cues that pattern the early postimplantation embryo, although almost nothing is known about these processes in humans. These possibilities and others discussed in this chapter are summarized in Table 3-1 .

Reasons for Laboratory Studies of Human Embryos.

Understanding of Human Development

Genome editing by CRISPR/Cas9 and similar techniques has a key place in the tool set needed to undertake such experiments. CRISPR/Cas9guided activation or inactivation of specific target pathways could be used to understand overall gene regulation in development. Indeed, as the efficiency of CRISPR/Cas9 continues to increase, it should be possible to use genome editing to knock out 4 genes in zygotes and study the effects directly in genetically altered embryos. None of these experiments would involve human pregnancies, so none could result in heritable germline modifications. They would all be in vitro experiments, with results being analyzed primarily at the blastocyst stage in the first 1-6 days of development.

In some cases, there could be interest in exploring the effects of altering specific genes at the next stages of human development, notably the early stages after the embryo would implant in a uterus. At present, culture of human embryos up to the stage just prior to germ-layer formation (at 14 days after fertilization or the formation of the “primitive streak”) is permitted in many countries. Improved culture systems that allow human embryos to develop in culture during the implantation period are being developed. Recent results suggest that these systems could be used to study the elaboration of extraembryonic structures and of the epiblast into an “embryonic disc”—processes that occur in humans in ways not found in mice ( Deglincerti et al., 2016 ; Shahbazi et al., 2016 ). These improved cell culture systems, combined with better ways of analyzing gene function using genome editing, can be expected to lead to better understanding of the fundamental processes of early human development. Already at least two research groups (in the United Kingdom and Sweden) have received regulatory permission to carry out CRISPR/Cas9 experiments on human embryos, aimed at addressing these kinds of fundamental biological questions.

Knowledge gained from such studies is expected to inform and improve IVF procedures and embryo implantation rates and reduce rates of miscarriage. Conversely, the same studies may lead to novel methods of contraception. Such research also should lead to better ways of establishing and maintaining stem cells from these early embryonic stages, which could facilitate efforts to derive cell types for studies and treatments of disease and traumatic injury. Knowledge gained from these laboratory studies using genome-editing methods in early human embryos should also provide information about the suitability of these methods for any eventual potential clinical use. That is, basic research can be expected to inform an understanding of the feasibility of making heritable, and preferably non-mosaic, changes in the genome (see Chapter 5 ). Because human embryos that can be used in research are a valuable and relatively scarce resource, it will be important to ensure that the most efficient methods are used for these laboratory studies of their basic biology. Thus, it is likely that in the course of this research, various technical issues associated with improving the use of genome-editing methods in human embryos will be addressed. Relevant questions include

• the type and form of genome-editing components to be introduced;
• whether to use Cas9 or an alternative nuclease;
• what method to use to introduce the genome-editing components—for example, as DNA, mRNA, protein, or ribonucleoprotein complex;
• whether to use single guide RNAs, pairs, or multiple guide RNAs as part of the editing machinery;
• the size of the DNA template and whether such a template is required;
• the optimal timing for genome editing, that is, whether information can be obtained by using two-cell embryos, whether it is necessary to use one-cell embryos, or whether it is best to introduce the reagents along with the sperm during in vitro fertilization;
• whether mosaicism can be tolerated, keeping in mind that it may be an advantage for certain experiments, as when cell fate is to be followed, but may need to be avoided in other cases, such as when investigating a gene whose product is a secreted protein; and
• how to test and improve modified versions of nucleases such as Cas9 or inhibitors of certain repair mechanisms (e.g., an effective inhibitor of nonhomologous end joining may be needed if the experiment demands homology-directed repair [ Howden et al., 2016 ]).

Understanding of Gametogenesis and Infertility

In mice, the generation of spermatogonial stem cell (SSC) lines from the adult testes has provided a rich source of cells with which to study the process of spermatogenesis in vitro and in vivo, after regrafting to the testes. It is possible to alter these cells genetically and study the impact of the changes on the process of spermatogenesis itself or, in mice, the impact on the offspring. It is also possible to correct genetic mutations in the stem cells in vitro using CRISPR/Cas9. Proof of principle for such an approach has been published ( Wu et al., 2015 ). This work used CRISPR/Cas9 editing in mouse SSCs to correct a gene mutation that causes cataracts in mice. The edited SSCs were transferred back to mouse testes, and round spermatids were collected for intracytoplasmic sperm injection (ICSI), a form of IVF, to create embryos. Resulting offspring were correctly edited at 100 percent efficiency. Similar experiments have been conducted using SSCs from other species, including macaques ( Hermann et al., 2012 ). Stable human SSC lines have not yet been reported, but would clearly be an important tool for understanding male infertility and for exploring such issues as the higher rate of mutations associated with age. This is an active area of research because it may enable restoration of fertility in male cancer patients after radiation or chemotherapy. The ability to grow and manipulate human SSCs would, however, raise the possibility of generating human germline alterations if the cells were grafted back to the testes or used in IVF.

Related issues arise from experiments in which both oocytes and sperm progenitors have been generated from mouse ES cells. ES-derived oocytes can be fertilized by normal sperm, and ES-derived spermatids can fertilize eggs by ICSI ( Hayashi et al., 2012 ; Hikabe et al., 2016 ; Saitou and Miyauchi, 2016 ; Zhou et al., 2016 ). Human gametes have not yet been generated successfully from pluripotent stem cells, although two recent papers report the generation of early germ cell progenitors from human ES cells ( Irie et al., 2015 ; Sasaki et al., 2015 ). Through the use of genome-editing methods, this work also highlighted significant differences between mice and humans in the genes involved in specification of primordial germ cells. There is evidence as well that knowledge gained from studying later stages of spermatogenesis in mice may not always be applicable to the same process in humans. These findings reflect the role of research on human cells in answering questions about human biology. If human haploid gametes could be generated from human pluripotent cells, as they can be in mice, it would open up new avenues for understanding gametogenesis and the causes of infertility. It would also open up possibilities for using heritable genome modifications to address health problems that originate from genetic causes.

• ETHICAL AND REGULATORY ISSUES IN BASIC RESEARCH

As described in more detail in Chapter 2 , basic science research performed in the laboratory on somatic cells will be subject to regulation focused on safety for laboratory workers and the environment, including special review by institutional biosafety committees for work involving recombinant DNA. Few new ethical issues are raised, although if the cells and tissues come from identifiable living individuals, donor consent and privacy will be a concern, and in most cases the protocols will be subject to at least some review by institutional review boards.

Research with embryos is more controversial. As noted earlier, research using viable embryos is illegal in a small number of U.S. states ( NCSL, 2016 ), and while permitted in most states, research that exposes embryos to risk generally may not be funded by the U.S. Department of Health and Human Services (HHS); this is due to the Dickey-Wicker Amendment, 5 which has been adopted repeatedly since the 1990s as part of the HHS appropriations process, including in the bills introduced for 2017 funding (see Chapter 2 ). 6 It states

(a) None of the funds made available in this Act may be used for— (1) the creation of a human embryo or embryos for research purposes; or (2) research in which a human embryo or embryos are destroyed, discarded, or knowingly subjected to risk of injury or death greater than that allowed for research on fetuses in utero under 45 CFR 46.204(b) and section 498(b) of the Public Health Service Act (42 U.S.C. 289g(b)). (b) For purposes of this section, the term “human embryo or embryos” includes any organism, not protected as a human subject under 45 CFR 46 as of the date of the enactment of this Act, that is derived by fertilization, parthenogenesis, cloning, or any other means from one or more human gametes or human diploid cells.

The effect of this combination of state and federal law is to make embryo research legal in most of the United States but generally not eligible for HHS funding.

Additional, extralegal oversight of laboratory research using human embryos comes from the stem cell research oversight committees that were widely adopted pursuant to recommendations of the National Academies regarding embryonic stem cell research ( IOM, 2005 ; NRC and IOM, 2010 ). Recently, the International Society for Stem Cell Research, whose membership includes investigators from around the world as well as the United States, adopted guidelines calling for the transformation of these voluntary stem cell research oversight committees into human embryo research oversight (EMRO) committees that would oversee “all research that (a) involves preimplantation stages of human development, human embryos, or embryo-derived cells or (b) entails the production of human gametes in vitro when such gametes are tested by fertilization or used for the creation of embryos” ( ISSCR, 2016a , p. 5). The review would include details of the proposal and the credentials of the researchers under the auspices of these independent, multidisciplinary committees of scientists, ethicists, and members of the public. The proposed committees would assess research goals “within an ethical framework to ensure that research proceeds in a transparent and responsible manner. The project proposal should include a discussion of alternative methods and provide a rationale for employing the requested human materials, including justification for the numbers of preimplantation embryos to be used, the proposed methodology, and for performing the experiments in a human rather than animal model system” ( ISSCR, 2016a , p. 6).

• CONCLUSIONS AND RECOMMENDATION

Laboratory research involving human genome editing—that is, research that does not involve contact with patients—follows regulatory pathways that are the same as those for other basic laboratory in vitro research with human tissues, and raises issues already managed under existing ethical norms and regulatory regimes. This includes not only work with somatic cells, but also the donation and use of human gametes and embryos for research purposes, where this research is permitted. While there are those who disagree with the policies embodied in some of those rules, the rules continue to be in effect. Important scientific and clinical issues relevant to human fertility and reproduction require continued laboratory research on human gametes and their progenitors, human embryos, and pluripotent stem cells. This research is necessary for medical and scientific purposes that are not directed at heritable genome editing, though it will also provide valuable information and techniques that could be applied if heritable genome editing were to be attempted in the future.

RECOMMENDATION 3-1. Existing regulatory infrastructure and processes for reviewing and evaluating basic laboratory genome-editing research with human cells and tissues should be used to evaluate future basic laboratory research on human genome editing.

SCNT is a technique in which the original nucleus of an egg cell is removed and replaced with a “donor” nucleus taken from another cell (e.g., from a somatic cell that has undergone genome editing). This is the technique that was used to create Dolly, the first cloned mammal obtained from an adult cell.

Having a chromosome number that is not an exact multiple of the usual haploid number.

The term “epigenome” refers to a set of chemical modifications to the DNA of the genome and to proteins and RNAs that bind to DNA in the chromosomes to affect whether and how genes are expressed.

A gene is said to be “knocked out” when it is inactivated because the original DNA sequence has either been replaced or disrupted.

Public Law No. 114-113, Division H, Title V, § 508.

§ 508(a) in both S. 3040 and H.R. 5926.

• Cite this Page National Academies of Sciences, Engineering, and Medicine; National Academy of Medicine; National Academy of Sciences; Committee on Human Gene Editing: Scientific, Medical, and Ethical Considerations. Human Genome Editing: Science, Ethics, and Governance. Washington (DC): National Academies Press (US); 2017 Feb 14. 3, Basic Research Using Genome Editing.
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WHO issues new recommendations on human genome editing for the advancement of public health

Two new companion reports released today by the World Health Organization (WHO) provide the first global recommendations to help establish human genome editing as a tool for public health, with an emphasis on safety, effectiveness and ethics.

The forward-looking new reports result from the first broad, global consultation looking at somatic, germline and heritable human genome editing. The consultation, which spanned over two years, involved hundreds of participants representing diverse perspectives from around the world, including scientists and researchers, patient groups, faith leaders and indigenous peoples.

“Human genome editing has the potential to advance our ability to treat and cure disease, but the full impact will only be realized if we deploy it for the benefit of all people, instead of fueling more health inequity between and within countries,” said Dr Tedros Adhanom Ghebreyesus, WHO Director-General.

Potential benefits of human genome editing include faster and more accurate diagnosis, more targeted treatments and prevention of genetic disorders. Somatic gene therapies, which involve modifying a patient’s DNA to treat or cure a disease, have been successfully used to address HIV, sickle-cell disease and transthyretin amyloidosis. The technique could also vastly improve treatment for a variety of cancers.

However, some risks exist, for example, with germline and heritable human genome editing, which alter the genome of human embryos and could be passed on to subsequent generations, modifying descendants’ traits.

The reports published today deliver recommendations on the governance and oversight of human genome editing in nine discrete areas, including human genome editing registries; international research and medical travel; illegal, unregistered, unethical or unsafe research; intellectual property; and education, engagement and empowerment. The recommendations focus on systems-level improvements needed to build capacity in all countries to ensure that human genome editing is used safely, effectively, and ethically.

The reports also provide a new governance framework that identifies specific tools, institutions and scenarios to illustrate practical challenges in implementing, regulating and overseeing research into the human genome. The governance framework offers concrete recommendations for dealing with specific scenarios such as:

• A hypothetical clinical trial of somatic human genome editing for sickle cell disease proposed to take place in West Africa
• Proposed use of somatic or epigenetic genome editing to enhance athletic performance
• An imaginary clinic based in a country with minimal oversight of heritable human genome editing that offers these services to international clients following in vitro fertilization and preimplantation genetic diagnosis

“These new reports from WHO’s Expert Advisory Committee represent a leap forward for this area of rapidly emerging science,” said WHO’s Chief Scientist, Dr Soumya Swaminathan. “As global research delves deeper into the human genome, we must minimize risks and leverage ways that science can drive better health for everyone, everywhere.”

What’s next

• Convene a small expert committee to consider next steps for the Registry, including how to better monitor clinical trials using human genome editing technologies of concern
• Convene multisector stakeholders to develop an accessible mechanism for confidential reporting of concerns about possibly illegal, unregistered, unethical and unsafe human genome editing research and other activities
• As part of a commitment to increase ‘education, engagement and empowerment’, lead regional webinars focusing on regional/local needs. Work within the Science Division to consider how to build an inclusive global dialogue on frontier technologies, including cross-UN working and the creation of web-based resources for reliable information on frontier technologies, including human genome editing. 

Meeting recording

Reports launch – Human Genome Editing: A Framework for Governance and Recommendations, July 14, 2021. 

Moderator: Professor John Reeder, Director Research for Health , WHO

Panellists: Members of the WHO Expert Advisory Committee on Developing Global Standards for Governance and Oversight of Human Genome Editing”

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World Health Organization

Human genome editing: recommendations

Human genome editing: a framework for governance

Human genome editing: position paper

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• Published: 03 January 2020

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

• Hongyi Li 1   na1 ,
• Yang Yang 1   na1 ,
• Weiqi Hong 2 ,
• Mengyuan Huang 2 ,
• Min Wu 3 &
• Xia Zhao 1  

Signal Transduction and Targeted Therapy volume  5 , Article number:  1 ( 2020 ) Cite this article

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• Gene therapy
• Genetic techniques

Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

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Precise genome-editing in human diseases: mechanisms, strategies and applications

Advances in CRISPR therapeutics

The promise and challenge of therapeutic genome editing

Introduction.

Over the last few years, the exuberant development of genome editing has revolutionized research on the human genome, which has enabled investigators to better understand the contribution of a single-gene product to a disease in an organism. In the 1970s, the development of genetic engineering (manipulation of DNA or RNA) established a novel frontier in genome editing. 1 Based on engineered or bacterial nucleases, genome editing technologies have been developed at a rapid pace over the past 10 years and have begun to show extraordinary utility in various fields, ranging from basic research to applied biotechnology and biomedical research. 2 Genome editing can be achieved in vitro or in vivo by delivering the editing machinery in situ, which powerfully adds, ablates and “corrects” genes as well as performs other highly targeted genomic modifications. 3 , 4 Targeted DNA alterations begin from the generation of nuclease-induced double-stranded breaks (DSBs), which leads to the stimulation of highly efficient recombination mechanisms of cellular DNA in mammalian cells. 5 , 6 Nuclease-induced DNA DSBs can be repaired by one of the two major mechanisms that occur in almost all cell types and organisms: homology-directed repair (HDR) and nonhomologous end-joining (NHEJ), 7 resulting in targeted integration or gene disruptions, respectively (Fig. 1 ).

Genome editing platforms and mechanisms for DSB repair with endogenous DNA. Genome editing nucleases (ZFNs, TALENs and CRISPR/Cas9) induce DSBs at targeted sites. DSBs can be repaired by NHEJ or, in the presence of donor template, by HDR. Gene disruption by targeting the locus with NHEJ leads to the formation of indels. When two DSBs target both sides of a pathogenic amplification or insertion, a therapeutic deletion of the intervening sequences can be created, leading to NHEJ gene correction. In the presence of a donor-corrected HDR template, HDR gene correction or gene addition induces a DSB at the desired locus. DSB double-stranded break, ZFN zinc-finger nuclease, TALEN transcription activator-like effector nuclease, CRISPR/Cas9 clustered regularly interspaced short palindromic repeat associated 9 nuclease, NHEJ nonhomologous end-joining, HDR homology-directed repair.

Historically, homologous recombination (HR), in which undamaged homologous DNA fragments are used as templates, has been the approach to realize targeted gene addition, replacement, or inactivation; however, the utility of HR is heavily limited due to its inefficiency in mammalian cells and model organisms. 8 After it was discovered that DSBs could raise the incidence of HDR by multiple orders of magnitude, targeted nucleases have been found as an alternative approach to increase the efficiency of HDR-mediated genetic alteration. Once a targeted DSB has been made, HDR may reconstruct the cleaved DNA using an exogenous DNA template analog to the break site sequence.

This mechanism may be used to introduce precise mutations by delivering an appropriately designed repair template into targeted cells directly, 9 , 10 thereby, in a site-specific manner, resulting in mutation correction or new sequence insertion. Alternatively, NHEJ-mediated repair tends to result in errors because it leads to efficient formation of gene insertion or deletion (indels) in diverse lengths at the DSB site, which eventually causes gene inactivation. 11 If indels occur in the coding sequence, there will be frameshift mutations, which will result in mRNA degradation or nonfunctional truncated protein production by nonsense-mediated decay. 12 This approach and its applications are thought to be simpler than HR-based methods because (a) there is no need for a repair matrix and (b) the cell type has less impact on modification efficacy (contrary to HR, NHEJ may be active all through the cell cycle). 13 Thus, similar to RNAi, NHEJ may be applied in immortalized cell lines to generate the inactivation of a single gene or multiple genes, but by creating loss-of-function mutations, it may lead to permanent gene inactivation. 9

In the early development stage of genome editing, to induce the desired DSBs at each particular DNA target site, the engineering of distinct zinc-finger nucleases (ZFNs) 14 or meganucleases 15 has been the research focus. These nuclease systems required specialized competence to generate artificial proteins consisting of customizable sequence-specific DNA-binding domains, each connected to a nonspecific nuclease for target cleavage, providing researchers with unprecedented tools to perform genetic manipulation. 16 Subsequently, a new class of a Flavobacterium okeanokoites (FokI) catalytic domain derived from bacterial proteins termed transcription activator-like effectors (TALEs) has shed light on new possibilities for precise genome editing. 17 TALE-based programmable nucleases can cleave any DNA sequence of interest with relatively high frequency. However, the main challenges for transcription activator-like effector nucleases (TALEN) approaches are the design of a complex molecular cloning for each new DNA target and its low efficiency of genome screening in successfully targeted cells. 18 Clustered regularly interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) nuclease is a recently discovered, robust gene editing platform derived from a bacterial adaptive immune defense system. 19 This system can be efficiently programmed to modify the genome of eukaryotic cells via an RNA-guided DNA cleavage module and has emerged as a potential alternative to ZFNs and TALENs to induce targeted genetic modifications 20 (Table 1 ). Since 2013, when it was first applied in mammalian cells as a tool to edit the genome, 21 , 22 the versatile CRISPR/Cas9 technology has been rapidly expanding its use in modulating gene expression, ranging from genomic sequence correction or alteration to epigenetic and transcriptional modifications.

The advent of programmable nucleases has greatly accelerated the proceedings of gene editing from concept to clinical practice and unprecedentedly enabled scientists with a powerful tool to maneuver literally any gene in a wide variety of cell types and species. Current preclinical research on genome editing primarily concentrates on viral infections, cardiovascular diseases (CVDs), metabolic disorders, primary defects of the immune system, hemophilia, muscular dystrophy, and development of T cell-based anticancer immunotherapies. Some of these methods have gone beyond preclinical research and are recently undergoing phase I/II clinical trials. Here, we review recent improvements of the three main genome editing platforms (ZFN, TALENs, and CRISPR/Cas9) and discuss applications of their derivative reagents as gene editing tools in various human diseases and in promising future therapies, focusing on eukaryotic cells and animal models. Finally, we outline the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

Structure and mechanism of genome editing tools

The structure of zfns and their interaction with dna.

ZFNs are assembled by fusing a non-sequence-specific cleavage domain to a site-specific DNA-binding domain that is loaded on the zinc finger. 23 The zinc-finger protein with site-specific binding properties to DNA was discovered primarily in 1985 as part of transcription factor IIIa in Xenopus oocytes. 24 The functional specificity of the designed zinc-finger domain comprises an array of Cys 2 His 2 zinc fingers (ZFs), which are derived by highly conserved interactions of their zinc-finger domains with homologous DNA sequences. Generally, an individual Cys 2 His 2 zinc finger consists of approximately 30 amino acids, which constitute two anti-parallel β sheets opposing an α-helix. 25 Cys 2 -His 2 -ZF is an adaptable DNA recognition domain and is considered to be the most common type of DNA-binding motif in eukaryotic transcription factors. 26 Each zinc-finger unit selectivity recognizes three base pairs (bp) of DNA and produces base-specific contacts through the interaction of its α-helix residues with the major groove of DNA. 27 , 28 The FokI type II restriction endonuclease forms the domain that cleaves the DNA, which can be adopted as a dimer to directly target sequences within the genome for effective gene editing. 29 Since the FokI nuclease needs to be dimerized to cleave DNA, two ZFN molecules are usually required to bind to the target site in an appropriate orientation, 30 doubled in the number of specifically recognized base pairs. After DNA cleavage by ZFNs is achieved in eukaryotic cells, DSBs at a specific locus of the genome is initiated, creating the desired alterations in subsequent endogenous NHEJ or HDR repair systems. 23

The target sequence recognition and specificity of ZFNs are determined by three major factors: (a) the amino acid sequence of each finger, (b) the number of fingers, and (c) the interaction of the nuclease domain. By virtue of the modular structure of ZFNs, both the DNA-binding and catalytic domains can be individually optimized, which enables scientists to develop novel modular assembly with sufficient affinity and specificity for genome engineering. In early studies, individual ZFNs containing 3–6 fingers were used to interact with a 9–18 nucleotide target, which enabled ZFN dimers to specify 18–36 bp of DNA at each cleavage site. 31 Since the 18 bp sequence of DNA can render specificity within 68 billion bp of DNA, this approach facilitated the targeting of specific sequences in the human genome for the first time. A more recently developed strategy used architectural diversification to improve the targeting accuracy of ZFNs via “selection-based methods” 32 : this study developed a new linker option for spanning finger–finger and finger–FokI cleavage domain junctions, which produced a 64-fold total increase in the number of ZFN configurations available for targeting cleavage to any given base of DNA.

TALENs: a protein-based DNA targeting system

TALENs are another type of engineered nuclease that exhibit better specificity and efficiency than ZFNs. Similar to ZFNs, TALENs comprise a nonspecific DNA cleavage domain fused to a customizable sequence-specific DNA-binding domain to generate DSBs. This DNA-binding domain consists of a highly conserved repeat sequence from transcription activator-like effector (TALE), which is a protein originally discovered in the phytopathogenic Xanthomonas bacteria that naturally alters the transcription of genes in host plant cells. 17 , 33 The binding of TALE to DNA is mediated by a central region that contains an array of 33- to 35-amino-acid sequence motifs. The amino acid sequence of each repeat is structurally similar, except for two hypervariable amino acids (the repeat variable di-residues or RVDs) at positions 12 and 13. 34 DNA-binding specificity is determined by RVDs, with ND specifically binding to C nucleotides, HN to A or G nucleotides, NH to G nucleotides, and NP to all nucleotides. 17 There is a one-to-one correspondence between RVDs and contiguous nucleotides in the target site, constituting a strikingly simple TALE–DNA recognition cipher. 35

Functional endonuclease FokI is factitiously fused to DNA-binding domains to create site-specific DSBs and thereby stimulate DNA recombination to achieve TALEN-induced targeted genomic modification. To cleave the two strands of the targeted DNA, the FokI cleavage domain must be dimerized. Hence, like zinc fingers, such a TALEN module is designed in pairs to bind opposing DNA target loci, with proper spacing (12–30 bp) between the two binding sites. 36 However, compared to zinc-finger proteins, there is no need to redesign the linkage between repeats constituting long arrays of TALEs, which function to target individual genomic sites. Following pioneering works on zinc-finger proteins, multiple effector domains have become accessible to support the fusion of TALE repeats for different genomic modification purposes, including nucleases, 37 transcriptional activators, 18 and site-specific recombinases. 38 Although their simpler cipher codes provide better simplicity in design than triplet-confined zinc-finger proteins, one of the primary technical hurdles for cloning repeat TALE arrays is the design of a large scale of identical repeat sequences. To address this limitation, a few strategies have been established to facilitate the fast assembly of custom TALE arrays, including “Golden Gate” molecular cloning, 39 high-throughput solid phase assembly, 40 , 41 and connection-independent cloning techniques. 42

CRISPR/Cas9: a versatile tool for genome editing

Early in 1987, clustered regularly interspersed short palindromic repeats (CRISPRs) were originally discovered in E. coli and later in many other bacteria species. 43 The function of the short repeat sequences remained unclear for many years before several studies in 2005 characterized their similarities to phage DNA, and subsequent experiments revealed that these sequences took part in bacterial and archaea adaptive immune defense against offending foreign DNA by inducing RNA-guided DNA cleavage. 44 , 45 , 46 Generally, the CRISPR‐Cas systems are divided into two classes based on the structural variation of the Cas genes and their organization style. 44 Specifically, class 1 CRISPR–Cas systems consist of multiprotein effector complexes, whereas class 2 systems comprise only a single effector protein; altogether, six CRISPR-Cas types and at least 29 subtypes have been reported, 47 , 48 and the list is rapidly expanding. The most frequently used subtype of CRISPR systems is the type II CRISPR/Cas9 system, which depends on a single Cas protein from Streptococcus pyogenes (SpCas9) targeting particular DNA sequences and is therefore an attractive gene editing tool. 49 Mechanistically, the CRISPR/Cas9 system comprises two components, a single-stranded guide RNA (sgRNA) and a Cas9 endonuclease. The sgRNA often contains a unique 20 base-pair (bp) sequence designed to complement the target DNA site in a sequence-specific manner, and this must be followed by a short DNA sequence upstream essential for the compatibility with the Cas9 protein used, which is termed the “protospacer adjacent motif” (PAM) of an “NGG” or “NAG”. 50 , 51 The sgRNA binds to the target sequence by Watson–Crick base pairing and Cas9 precisely cleaves the DNA to generate a DSB. 52 Following the DSB, DNA-DSB repair mechanisms initiate genome repair. With the CRISPR/Cas9 system, through pathways of NHEJ or high-fidelity HDR, targeted genomic modifications, including the introduction of small insertions and deletions (indels), can be made. 53

Known as the RNA‐guided system, CRISPR/Cas9 is more suitable for application compared to other gene editing technologies and has several important advantages. 20 For example, endonuclease-based ZFN or TALEN tools demand reengineering of the enzyme to fit each target sequence, and they should be synthesized separately for each case; however, the nuclease protein Cas9 is identical in all cases and can be conveniently engineered to recognize new sites via changing the guide RNA sequences (sgRNA), which match target sites by Watson–Crick base pairing. Moreover, in contrast to CRISPR/Cas9, ZFNs and TALENs demand much more labor and are more expensive. An additional advantage of CRISPR/Cas9 is that it has the potential of simultaneous multiple loci editing, making this technology easier, more efficient, and more scalable compared to other genome editing technologies. CRISPR/Cas9 is now an indispensable tool in biological research.

Three common strategies have been developed for genome editing with the CRISPR/Cas9 platform: (1) the plasmid‐based CRISPR/Cas9 strategy, where a plasmid is used to encode Cas9 protein and sgRNA, 21 , 22 assembles Cas9 gene as well as sgRNA into the same plasmid in vitro. this strategy is longer lasting in the expression of Cas9 and sgRNA, and it prevents multiple transfections. 54 However, the encoded plasmid needs to be introduced inside the nucleus of target cells, which is a key challenge in this system; (2) direct intracellular delivery of Cas9 messenger RNA (mRNA) and sgRNA, 55 the greatest drawback of which lies in the poor stability of mRNA, which results in transient expression of mRNA and a short duration of gene modification; (3) directly delivery of Cas9 protein and sgRNA 56 , which has several advantages, including rapid action, great stability, and limited antigenicity.

The editing of DNA means the irreversible permanent change of genome information, and this process is also facing inevitable security risks and ethical problems. In addition, some cell types, such as neurons, are difficult to modify DNA using CRISPR/Cas9-mediated editing, which limits the use of gene therapy for nervous system diseases. As a result, genome editing strategies that only edit and modify RNA have also been proposed by scientists. 57 , 58 As an intermediate product of DNA transcription, RNA is responsible for guiding the production of downstream proteins. With the use of CRISPR technology, RNA mutation is modified briefly, which not only avoids the irreversible modification of the genome but also can repair protein function in almost all cells to treat a variety of diseases. Stem cell transplantation combined with the CRISPR/Cas9 system is another approach for the therapy of genetic mutations. It has been proven that patient-induced pluripotent stem cells (iPSCs) have the ability to differentiate into retinal precursors, and it is a useful cell source for cell replacement therapy without immune rejection problems. 59 , 60 However, patient-derived iPSCs might still harbor the same pathogenic genes, which could influence the therapeutic efficacy of transplanted cells. Therefore, it is necessary to combine the CRISPR/Cas9 system to fix disease-causing mutations in patient-derived iPSCs before transplantation. 61

Genome editing for disease modeling and gene therapy

Targeted gene modification via chimeric genome editing tools (e.g., ZFNs, TALENs, and CRISPR/Cas9) is a powerful method to assess gene function and precisely manipulate cellular behavior and function. These genome editing tools have enabled investigators to use genetically engineered animals to understand the etiology behind various diseases and to clarify molecular mechanisms that can be exploited for better therapeutic strategies (Fig. 2 ).

Ex vivo and in vivo genome editing for clinical therapy. Right: For in ex vivo editing therapy, cells are isolated from a patient to be treated, edited and then re-engrafted back to the patient. To achieve therapeutic success, the target cells must be able to survive in vitro and return to the target tissue after transplantation. Left: For in vivo editing therapy, engineered nucleases are delivered by viral or nonviral approaches and directly injected into the patient for systemic or targeted tissue (such as the eye, brain, or muscle) effect.

Cancer research

Oncogenes and mutant tumor suppressor genes provide outstanding opportunities for the use of genome modulating approaches. 62 Genome editing technology has accomplished crucial targeted cleavage events in various fundamental studies, from its initial proofs of efficient gene editing in eukaryotes to its recent applications in the engineering of hematopoietic stem cells (HSCs) and tumor-targeted T cells; this technology has established novel concepts of gene modification and has extended to a border field of cancer research.

As an archetypal platform for programmable DNA cleavage, ZFN-mediated targeting has been successfully applied to modify many genes in human cells and a number of model organisms, thus opening the door to the development and application of genome editing technologies. ZFN-driven gene disruption was primarily demonstrated in 1994 when a three-finger protein was constructed to specifically block the expression of the BCR-ABL human oncogene that was transformed into a mouse cell line. 63 After that, a study used a human lymphoblast cell line derived from chronic myeloid leukemia (CML) patients, and a custom-designed ZFN was applied to this cell line to deliver site-specific DSBs to the telomeric portion of the mixed lineage leukemia (MLL) gene breakpoint cluster region as well as to analyze chromosomal rearrangements associated with MLL leukemogenesis via DSB error repair. 64 Successful targeted modulation was also achieved using designed ZFNs, which promoted the disruption of endogenous T cell receptor (TCR) β- and α-chain genes. Lymphocytes treated with ZFNs lacked the surface expression of CD3-TCR and expanded with an increase in interleukin-7 (IL-7) and IL-15. 65 By targeting the promoter function of long terminal repeat (LTR) from human T cell leukemia virus type 1 (HTLV-1), a novel therapeutic ZFN specifically killed HTLV-1-infected cells in an in vivo model of adult T cell leukemia (ATL). 66 In addition, it was reported that effective cleavage of the BCR-ABL fusion gene by highly specific ZFNs terminated the translation of the BCR-ABL protein and induced apoptosis in imatinib-resistant CML cells. 67 Furthermore, cancer-relevant translocations in human Ewing sarcoma and anaplastic large cell lymphoma (ALCL) cells induced by ZFNs demonstrated that precise genomic rearrangements can be achieved in relevant cell types by custom nucleases. 68 Furthermore, the use of HER2-positive cell-penetrating peptide (CPP) conjugated to mammalian mTOR-specific ZFN made the mTOR locus nonfunctional and inhibited relevant cancer signaling pathways, providing insight into the design of novel molecular targeted therapeutics for breast cancer (in particular) and other types of cancers. 69 Moreover, as the tumor suppressor gene p53 plays a pivotal role in preventing cancer development, strategies of genome editing to restore wild-type p53 function have been investigated. A yeast-one-hybrid (Y1H) four-finger ZFN was designed to replace mutant p53 with wild-type p53 in several cancer cell lines (from glioblastoma, leukemia and breast cancer) via ZFN-induced HR. 70 Although the HR events were not particularly effective in this case, modifications at p53 loci still provided a framework for further investigation. In addition to modifying viral genes associated with tumorigenesis, researchers have applied ZFNs to optimize T cell-mediated antitumor therapy. For example, by importing a chimeric TCR that comprises an extracellular IL-13 domain (zetakine) and a cytoplasmic CD3 domain into CD8 + T cells, glioblastoma-specific cytolytic T lymphocytes (CTLs) can be generated. To achieve this goal, Reik et al. 71 knocked down the glucocorticoid receptor in the modified CTLs with ZFNs. Consequently, the cytolytic activity of “zetakine” transgenic CTLs against glioblastomas was preserved regardless of the presence of glucocorticoid treatment. This technology has recently been effective in knocking out glucose transport-related genes (MCT4 or BSG) in two glycolytic tumor models: colon adenocarcinoma and glioblastoma. 72

A milestone of TALENs was achieved when they were primarily applied to efficiently disrupt the endogenous genes NTF3 and CCR5 in human leukemia cells via the introduction of NHEJ- or HDR-induced modification into a coding sequence, demonstrating that TALENs could be designed for selective endogenous gene cleavage. 73 Interestingly, when TALENs and ZFNs were compared abreast at two human loci (CCR5 and IL2RG), TALENs showed a significant reduction in cytotoxicity. Moreover, the CCR5-specific TALEN was able to distinguish between the CCR5 target locus and a highly similar site in CCR2 when compared with ZFN technology. 37 By adopting TALEN gene editing technology, precise disruptions have also been introduced into the T cell receptor α constant (TRAC) gene and the CD52 gene in allogeneic T cells by TALEN-induced HDR. The TALEN used in this study was engineered by a retroviral vector that expressed a chimeric antigen receptor (CAR) targeting CD19+ leukemic B cells, which helped to develop the “universal” CAR T cells (dKO-CART19). 74 Alternatively, a site-specific TALEN was used to disrupt a single allele of the Fms-related tyrosine kinase 3 (FLT3) gene and generate isogenic leukemia cell clones. TALEN-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These suppressive effects were maintained in vivo and improved the survival rate of NOD/SCID mice transplanted with mutant K562 clones. 75 The use of engineered TALENs in prostate cancer cells functionally classifies androgen receptor (AR) target gene rearrangements as drivers of resistance. 76 Using TALENs to precisely cut the relevant translocation breakpoints, Piganeau et al. induced cancer-relevant translocations in anaplastic large cell lymphoma (ALCL). 68 Through an analogous strategy, the reversion of ALCL translocation was achieved in a patient cell line, restoring the integrity of the two involved chromosomes. Recent studies have also shown that TALEN gene editing technology used to knock out genes in cancer cells (including cells from prostate cancer, 76 breast cancer, 77 and hepatocellular carcinoma (HCC) 78 ) is a powerful and broadly applicable platform to explore gene mutations at the molecular level.

Because of its multiple advantages in genome editing, the CRISPR/Cas9 system has attracted considerable attention, and scientists gradually consider it to be a powerful therapeutic tool for treating diseases associated with genome mutations. The ultimate goal of cancer therapy with CRISPR/Cas9 is to remove malignant mutations and replace them with normal DNA sequences. 79 In a recent study, the leukemia model was generated by reviving several inactivated oncogenes through the lentiviral delivery of the Cas9-sgRNA system in primary hematopoietic stem and progenitor cells (HSPCs). 80 In this study, the pooled lentiviruses targeted genes, including Tet2, Runx1, Dnmt3a, Nf1, Ezh2, and Smc3. The objective HSPCs were selected via a fluorescent marker; those HSPCs are engaged in the development of myeloid neoplasia. CRISPR/Cas9 technology has also been adopted to establish organoid tumor models. 81 , 82 For instance, organoid colon cancer models were constructed in vitro with CRISPR technology by introducing mutations of tumor suppressor genes (APC, TP53, SMAD4, etc.) and gene modification of oncogenes (KRAS, PI3K, etc.). 83 Moreover, guided by colonoscopy, through mucosal injection, Roper et al. 84 established CRISPR engineered mouse tumor organoids by delivering viral vectors carrying CRISPR/Cas9 components to the distal colon of mice. Such an approach has already been applied in a study modeling tumor progression with an adenoma-carcinoma-metastasis sequence. In the future, the use of CRISPR/Cas9 technology to establish precise cancer models will significantly promote the research of functional cancer genomics and facilitate the advancement of cancer therapies.

Cardiovascular disease

CVD is a serious hazard to human health and is the number one cause of death in many industrialized countries. Many different types of CVD are usually associated with a single genetic mutation or a combination of rare inherited heterozygous mutations. 85 In practice, clinical treatments focus on the relief of disease symptoms without addressing potential genetic defects. Currently, the establishment of in vivo CVD models with gene editing technology and the in-depth analysis of CVD pathogenic genes as well as their molecular mechanisms have made it possible to test the ability of gene therapy to control specific gene expression and improve gene functions. With the help of genome editing technologies, various research models of cardiovascular conditions have been created.

Abrahimi et al. 86 used CRISPR/Cas9 to efficiently ablate major histocompatibility complex class II (MHCII) with double gene knockout in normal human endothelial cells. These cells retain the ability to form vascular structures without activating allogeneic CD4+ T cells. It is promising to apply such technology in the field of allograft bioengineering, including the refinement of heart transplant. In addition, CRISPR/Cas9 technology can accurately remove β2M and CCR5 on CD34+ HSCs while retaining its ability to undergo multidifferentiation, which provides the possibility for the future treatment of ischemic heart conditions with HSCs. 86 In another study, Carroll et al. 87 established a cardiac-specific transgenic mouse model by injecting Cas9-containing plasmids into mouse zygotes; the expression of Cas9 was regulated by the Myh6 promoter. In this transgenic model, high levels of Cas9 were expressed exclusively in heart cardiomyocytes. The investigators then intraperitoneally injected sgRNA targeting Myh6 loaded in an adeno-associated virus (AAV) vector, subsequently inducing cardiac-specific gene modification at the Myh6 locus, finally leading to hypertrophic cardiomyopathy.

It has been demonstrated in the whole-exome sequencing of a nuclear family that three missense variants of a single nucleotide in the MKL2, MYH7, and NKX2-5 genes pass on to three offspring with cardiomyopathy with childhood onset. 88 Gifford et al. 89 adopted CRISPR/Cas9 to establish a mouse model that encodes orthologous variants and showed that the complex of heterozygosity of all three variants reproduced the phenotype of human disease. An analysis of mouse heart and human induced pluripotent stem-cell-derived cardiomyocytes provides histological and molecular evidence for the contribution of the NKX2-5 variant as a genetic modifier.

Porcine models resemble human conditions by physiology, anatomy, and genetics and are often considered ideal models for human cardiovascular structure research. Yang et al. 90 applied ZFN technology with nuclear transfer in somatic cells to generate endogenous gene knockout pigs, which have a specific mutation in peroxisome proliferator-activated receptor gamma (PARP-γ). Marfan syndrome (MFS) is an autosomal dominant disease caused by a mutation of heterozygous fibrillin-1 (FBN1) and presents cardiovascular symptoms and skeletal abnormalities. By the same principle, Umeyama et al. 91 accomplished the establishment of FBN1 mutant cloned pigs (+Glu433AsnfsX98), which exhibited phenotypes similar to those of humans with MFS, such as scoliosis, funnel chest, delayed epiphysis mineralization, and the destruction of elastic fiber structure in the medial aortic tissue.

Human induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology can also be combined to generate a congenital heart disease model associated with GATA4 mutations in vitro to investigate the pathogenesis of this gene mutation. 92 , 93 Using Barth syndrome (BTHS) iPSC-derived cardiomyocytes (iPSC-CMs) and genome editing, Wang and colleagues demonstrated that TAZ mutation is associated with myocardial metabolism and structural and functional abnormalities. 93 These findings indicate the value of genetically edited animals as models for research on the pathogenesis of CVD and provide new insights into treatment strategies.

By genome editing techniques, potential therapeutic methods of repairing disease-causing mutations or of knocking out specific genes as CVD prevention approaches have also received widespread attention. For example, long QT syndrome (LQTS) is an autosomal dominant congenital heart disease. Hybrid mutations in multiple genes may lead to LQTS, some of which have relatively clear mutation sites with known molecular functions, such as hERG gene mutations in the pore-forming subunit alpha protein that encodes the potassium voltage-gated channel. The hERG gene mainly expresses and functions in cells of the myocardium and smooth muscle, and its mutation can cause fatal ventricular arrhythmia. 94 Repairing hERG gene mutations in cardiomyocytes using CRISPR technology may be an effective strategy to treat such LQTS.

Previous studies have noted that nonsense mutation carriers of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have significantly decreased levels of low-density lipoprotein cholesterol (LDL-C) in their blood compared with normal subjects (an allelic mutation corresponds to a 30 to 40% reduction). 95 The blood level of triglyceride (TG) in subjects with nonsense mutations in the apolipoprotein C3 (APOC3) gene was significantly lower than that in unaffected people (an allelic mutation corresponds to a 40% decrease). 96 The incidence of heart disease in both carriers was lower than that in unaffected subjects by more than 80%, suggesting that the inhibition of PCSK9 and APOC3 gene expression can be used as a potential treatment for cardiovascular disease. Since these two genes are mainly expressed in liver cells, one idea is to directly introduce nonsense mutations to APOC3 or PCSK9 genes in liver cells through genome editing technology, thus fundamentally inhibiting protein synthesis and achieving long-term stable therapeutic effects. 97 , 98

PRKAG2 cardiac syndrome is an autosomal dominant disease induced by a mutation in the PRKAG2 gene encoding the AMP-activated protein kinase γ2 regulatory subunit. A recent study suggests that the selective destruction of pathogenic mutations through CRISPR/Cas9 technology in vivo is a competent strategy to treat PRKAG2 heart syndrome and other dominant hereditary heart conditions. 99

Metabolic diseases

Metabolic diseases refer to the pathological state in which the body’s protein, fat, carbohydrates, etc. are metabolically disordered. Metabolic diseases include a group of syndromes that are caused by both genetic factors and the environment. 100 Gene editing technology can be applied in functional gene screening, gene therapy and the construction of metabolic disease models, such as obesity, diabetes, and hyperlipidemia. Leptin (Lep) is a hormone secreted by white fat cells that acts on the metabolic regulation center of the hypothalamus through the leptin receptor (LepR). 101 It has diverse functions, including appetite suppression, energy intake reduction, and fat synthesis inhibition, and can regulate blood sugar concentration, neuroendocrine, etc. A number of animal models have been developed to illustrate the important role of Lep/LepR in glycolipid metabolism, and the most widely used are ob/ob mice against Lep and db/db mice against LepR. 102 Chen and colleagues injected TALEN components into rat zygotes to specifically knockout LepR, thus obtaining three lines of rats with LepR mutations. 103 Phenotypes in these strains manifested as obesity and other metabolic disorders; additionally, the authors established a LepR mutant obese rat model, exhibiting efficient germline transmission. Bao et al. 104 successfully established LepR knockout mice using CRISPR/Cas9 technology. Homozygous LepR-deficient mice are characterized by obesity, hyperphagia, hyperglycemia, insulin resistance, and lipid metabolism disorders, together with some complications of diabetes. The same principle has been used to generate the cytochrome P450 (CYP) 2E1 knockout rat model with CRISPR/Cas9 technology to explore the role of the CYP2E1 gene in biochemical metabolism, toxicology, and diseases (e.g., diabetes and alcoholic cirrhosis). 105 The FTO allele is associated with obesity, which inhibits the mitochondrial thermogenic effects in adipose precursor cells. FTO gene mutations inhibit the conversion of white fat to brown fat. The FTO gene-regulated thermogenic pathway involves ARID5B, rs1421085, IRX3, and IRX5 factors. rs1421085 can be edited using the CRISPR/Cas9 platform to repair the pattern structure of ARID5B, thereby suppressing the expression of IRX3 and IRX5 and achieving the effect of weight loss. 106

As an important “experimental tool”, the animal model of diabetes can be used for pathological observation, preclinical experiments and drug screening. In a study based on CRISPR/Cas9 technology, pX330 (containing gRNA and Cas9 sequences together with the donor DNA plasmid) was injected into the oocyte to generate new Cre tool mice and achieve the genetic manipulation of pancreatic β cells. 107 The Ins1 (insulin gene) promoter and stop codon sequences served as targets for recombinase Cre insertion. Progeny F1 mice were histologically labeled as Cre-loxP recombination, which was observed in all islets expressing insulin-positive cells and negatively expressed in other tissues. There was no significant difference in glucose tolerance between these genetically edited mice and wild-type mice. Applying CRISPR/Cas9 technology in human iPSCs to target diabetes-related genes has become a promising approach to explore the molecular mechanisms of diabetes. For example, human iPSCs are isolated from single-gene diabetic MODY patients, and possible mutations in genes such as HNF4A, GCK, PDX-1, and INS are edited by CRISPR; the edited iPSCs then differentiate into pancreatic progenitor cells and are later transplanted into patients. 108 In addition, gene editing tools can also structurally modify proteins that promote chromatin structural variation, such as methylase, demethylase, acetylase or deacetylase, to treat diabetes epigenetically. 109

Gene editing technology is also critically involved in the study of lipid metabolism. 110 cAMP responsive element binding protein 3-like 3 (CREB3L3), a transcription factor expressed in the liver and small intestine, controls the energy metabolic equilibrium in fasting response. Nakagawa et al. 111 used the one-step CRISPR/Cas9 system to establish the CREB3L3-floxed murine model for the first time and subsequently obtained mice that were knocked out of the CREB3L3 gene in the small intestine and liver, respectively. The evidence above provides a new understanding of the role of CREB3L3 in plasma triglyceride metabolism and its contribution to liver and intestinal cholesterol metabolism. Familial hypercholesterolemia is an autosomal single-gene dominant disease correlated with a defect in the low-density lipoprotein receptor (LDLR) gene, which causes a disorder of the body’s lipid metabolism. In 2012, Carlson et al. 112 used TALEN technology to target LDLR in porcine fetal fibroblasts and obtained miniature swine containing mono- and biallelic mutations in LDLR, thus generating models of familial hypercholesterolemia, which came with critical biomedical significance in simulating lipid metabolic syndrome. Recent genome-wide association studies have identified tribble homolog 1 (TRIB1) to be associated with lipoprotein metabolism in human hepatocytes. Hepatic-specific overexpression of Trib1 reduced plasma TG and cholesterol levels by reducing the production of VLDL; in contrast, Trib1-knockout mice showed elevated plasma TG and cholesterol levels due to the increased production of VLDL. 113 To further explore its regulation of lipid metabolism, Nagiec et al. 114 induced the destruction of the chromosome at the TRIB1 locus by delivering the CRISPR/Cas9 system into mouse liver via a nonpathogenic AAV, which increased the transcription of PCKS9 and the secretion of PCKS9 protein; these responses ultimately reduced the level of liver LDL receptors and increased the level of LDL-C in the blood.

Neurodegenerative diseases

Neurodegenerative diseases (NDs), at least including Huntington’s disease (HD), Alzheimer’s disease (AD), and Parkinson’s disease (PD), are a group of conditions that have attracted the most concern because there have been no specific diagnostic approaches or established treatments for them. 115 , 116 There are a few potential pathogenic mechanisms behind NDs, including the accumulation of proteins with abnormal structures, 117 impaired ubiquitin-proteasome and/or autophagic lysosomal pathways, 118 oxidative stress 119 and circuit alternations 120 , etc. These mechanisms indicate that NDs are induced by complicated interactions of multiple genetic factors; either alone or in combination, the interactions lead to clinical features. The emergence of gene editing platforms provides a convenient approach to study gene functions related to NDs. 121

In HD, in vitro investigations demonstrated that via ZFNs, chromosomal expression of the mutant huntingtin (HTT) gene was significantly reduced at both the protein and mRNA levels; in vivo studies revealed that via striatal AAV delivery into the HD R6/2 mice, ZFNs extensively suppressed cerebral expression of the HTT gene and ameliorated HD-related symptoms. 122 Additionally, the HTT exon 1 in human iPSCs derived from fibroblasts of HD patients (HD-iPSCs) can be corrected by TALENs. 123 , 124 To better understand the pathogenesis of HD, Yan et al. 125 adopted CRISPR/Cas9 to establish a genome-edited porcine model of HD in 2018, which internally expressed full-length mutant HTT. As a promising breakthrough in the field of NDs, the development of HTT gene knock-in pigs would be of great significance for pathogenesis research and therapy exploration in Huntington disease.

Mutations in the gene encoding amyloid precursor protein (APP) cause familial AD with nearly complete penetrance. 126 Mouse fibroblast cells overexpress APP by receiving electroporated ZFNs designed with a DNA fragment containing the promoter and the protein coding regions of APP. These transgenic cells can be used to elucidate aspects of the molecular mechanisms of AD pathogenesis, particularly those involved in the mutant amyloidogenic pathway affecting the APP coding sequence. 127 The A673V variant near the APP β-secretase cleavage site contributes to AD pathology by increasing Aβ and enhancing its aggregation as well as toxicity; 128 by contrast, the A673T variant, which is adjacent to the aspartyl protease β-site in APP, provides protection against AD progression. 129 When A673V and A673T were induced in normal iPSCs by TALEN technology, these cells differentiated and formed cortical neurons, presenting with different levels of AD-associated biomarkers. 130 In addition, through a gene editing platform based on single-stranded oligonucleotide DNA nucleotides and CRISPR/CAS-blocking mutations, Paquet et al. 131 generated human iPSCs with dominant AD-causing mutations in APP and presenilin 1 (PSEN1), both heterozygous and homozygous, leading to early disease onset; thereby, they yielded cortical neurons, which showed genotype-dependent phenotypes associated with AD. Apolipoprotein E4 (APOE4) is a genetic risk factor for late-onset AD, while ApoE2, which differs from APOE4 by only two bases (two C bases in APOE4, corresponding to two U bases in APOE2), is not a risk factor for AD. Zhang and his team introduced APOE4 RNA related to disease risk into cells and successfully changed the APOE4 to APOE2 sequence through the RESCUE (RNA Editing for Specific C to U Exchange) editing system by changing two C bases in APOE4, which is equivalent to converting the disease risk of the AD high-risk population carrying the APOE4 gene to zero. 132

Alpha-synuclein (SNCA) and leucine-rich repeat kinase 2 (LRRK2) are associated with autosomal dominant PD, whereas another group of genes are associated with autosomal-recessive PD, including parkin, phosphatase and tensin homolog–induced kinase 1 (PINK1), DJ-1, and ATPase type 13A2 (ATP13A2). 133 The missense mutation of SNCA and LRRK2 genes can be corrected by ZFNs in vitro. After correction, the mtDNA damage disappeared in differentiated neural progenitor and neural cells derived from iPSCs. 134 , 135 Additionally, Soldner et al. 136 combined genome-wide epigenetic information with CRISPR/Cas9 genome editing to generate a genetically precisely controlled experimental system in human iPSCs. This system has identified PD-associated risk variants in noncoding distal enhancer elements that regulate SNCA expression; it has also confirmed that the transcriptional disorder of SNCA is related to sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1. These results suggest that gene editing techniques can generate specific ND animal models for further exploration into human diseases, and they are potentially capable of offering a robust therapeutic approach against multiple human genetic defects that have been considered incurable.

Viral diseases

Gene editing platforms have emerged recently as antiviral therapeutics for treating infectious diseases, either by altering the host genes required by the virus or by targeting the viral genes necessary for replication. 137 To date, genome editing-based HIV therapy has involved modifying infection-related genes to produce HIV-resistant CD4+ T cells and subsequently reinfusing the edited cells into patients. In 2008, the anti-HIV efficacy of the ZFN system was first presented in preclinical studies by adopting primary human CD4+ T cells. 138 Approximately 50% of the CCR5 alleles were disrupted with ZFN, which was delivered by the chimeric Ad5/F35 adenoviral vector. HIV-infected mice transfused with ZFN-modified CD4+ T cells also better preserved their original CD4+ T cells and had lower viral loads than nontransfused mice. In 2009, a patient was functionally cured of HIV infection by transplanting allogeneic stem cells from a donor with a homozygous CCR5 d32 allele, 139 suggesting that it is feasible to obtain resistance to HIV by mimicking natural homozygous CCR5 d32 mutations with genome editing technologies. In addition, engineering CD34+ HSPCs instead of CD4+ T cells with the CCR5 ZFN pair provides a durable source of modified cells and protects the CD4+ myeloid cells that are susceptible to HIV-1 as well. 140 Further in vivo experiments showed that mice transplanted with ZFN-modified HSPCs experienced rapid selection for CCR5(-/-) cells, which had obviously lower levels of HIV-1 than the control group and maintained human cells throughout their tissues. The disruption of C-X-C chemokine receptor 4 (CXCR4) is also under exploration as a strategy for patients who harbor CXCR4-tropic HIV-1. 141 Simultaneous genetic inactivation of both CCR5 and CXCR4 in human CD4+ T cells by ZFNs confers protection against viruses that exclusively use the targeted coreceptor. 142 Nuclease platforms based on TALEN 143 and CRISPR/Cas9 144 , 145 , 146 are also being applied to disrupt CCR5 in T cells and HSPCs. Laboratory results from Ebina and Hu et al. 144 , 147 showed that CRISPR/Cas9 not only could specifically eradicate latent HIV infection but also could prevent new HIV infection. Similarly, Hendel et al. 146 recently demonstrated that the codelivery of chemically modified CCR5 sgRNA with Cas9 mRNA/protein enhanced the genome editing efficiency of human primary CD4+ T cells and CD34+ HSPCs, with no DNA delivery-associated toxicity.

The sustained expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7 is implicated in malignant transformation and is strongly associated with cervical cancer. 148 The targeted mutagenesis of those high-risk HPV genes by gene editing tools may be a potential genetic therapy and may reverse cervical cancer in situ. Ding et al. 149 constructed a ZFN that could specifically recognize and cleave HPV16/18 E7 DNA. In their study, ZFN-mediated HPV16/18 E7 DNA disruption directly decreased the expression of E7, which resulted in efficient growth inhibition and type-specific apoptosis in HPV16/18-positive cervical cancer cells in vitro. When different plasmid-encoded zinc-finger modules were introduced in vivo, the therapeutic effects of ZFNs were further confirmed, inhibiting tumor growth in mice bearing cervical cancer cells. Similar results in another study showed that using ZFNs to target HPV E7 induced specific shear of the E7 gene and attenuated its malignant biological effect. 150 Wayengera et al. 151 computationally generated paired zinc-finger arrays (pZFAs) to target and cleave the genomic DNA of HPV-type 16 and 18, respectively. The authors highlighted the therapeutic effect of ZFN-mediated gene disruption in HPV 16/18, which was achieved when HPV-derived viral plasmids or vectors were introduced into precancerous lesions to realize targeted mutagenesis and gene-therapeutic reversal of cervical neoplasia. Additionally, the combined treatment of ZFNs with two chemotherapeutic drugs (cisplatin and trichostatin A) increased the apoptotic rate by approximately two times more than that of ZFNs used alone in HPV16/18-positive cervical cancer cells. Both chemotherapeutic drugs coordinated with ZFNs to downregulate HPV16/18 E7 expression while elevating retinoblastoma 1 (RB1) expression. 150 TALEN-mediated targeting of HPV oncogenes E6 and E7 within host DNA resulted in restoration of the host tumor suppressors p53 and RB1, which not only reduced tumorigenicity in HPV-positive cell lines but also ameliorated HPV-related cervical malignancy in transgenic mouse models. 152 Furthermore, CRISPR‐Cas9/HPV16 E6/E7 sensitized cervical cancer cells to cisplatin, indicating the potential of application in cervical cancer therapy. 153

Hepatitis B virus (HBV) is the most important pathogen of liver disease. Cotransfection of engineered ZFN pairs with a target plasmid containing the HBV genome results in specific cleavage. 154 Rananan et al. 155 designed and screened an efficient gRNA targeting the HBV genomic locus and transmitted the sgRNA/Cas9 system by lentiviral vector to HepG2 cells that were integrated with HBV. Finally, the amount of covalently closed circular DNA (cccDNA) gradually decreased, dropping by 92% on the 36th day; HBV gene expression and replication were also inhibited. One study also attempted to knock out Epstein–Barr virus (EBV)-related genes using CRISPR/cas9 technology to treat latent infections caused by EBV. 156 They used a plasmid containing CRISPR/cas9 to treat Raji cells isolated from Burkitt’s lymphoma with EBV latent infection; then, they found that cell proliferation was significantly inhibited and intracellular EBV load was significantly reduced.

Genomic editing technology allows us to gain a deeper understanding of the mechanisms underlying variant diseases associated with viral infection and demonstrates tremendous potential in the development of therapeutic approaches against viral infections, which represent some of the most intractable diseases.

Hereditary eye diseases

In recent years, with the advancement of gene sequencing technology, it is more explicit to make the genetic diagnosis of a variety of hereditary eye diseases, such as congenital cataract, congenital glaucoma, retinitis pigmentosa (RP), congenital corneal dystrophy, Leber congenital amaurosis (LCA), retinoblastoma (RB), and Usher syndrome. 157

CRISPR/Cas9 has already been used to generate animal models of RP. Receptor expression enhancer protein 6 (REEP6), a member of the REEP/Yop1 family of proteins, influences the structure of the endoplasmic reticulum. 158 Arno et al. reported that biallelic mutations in REEP6 cause autosomal-recessive retinitis pigmentosa. 159 They identified variants in REEP6 in patients with RP from unrelated families. Moreover, they created a knock-in mouse model of Reep6 p.Leu135Pro via CRISPR/Cas9. The clinical phenotypes of RP were replicated in the Reep6L135P/L135P homozygous knock-in mice, such as developing photoreceptor degeneration and dysfunction of the rod photoreceptors, which provides a better animal model for future studies of RP. The rodless (rd1) mouse, the most vastly used preclinical model of RP, has been aggressively debated for nearly a century after its occurrence because the cause of the blinding RP phenotype remains undetermined. The rd1 mouse has two homozygous variants in the Pde6b locus of chromosome 5: a nonsense mutation (Y347X) and a murine leukemia virus (Xmv-28) insertion in the reverse orientation in intron 1. 160 , 161 Wu et al. repaired the nonsense point mutation via CRISPR/Cas9 to rescue and ameliorate the disease, demonstrating that the Y347X mutation in rd1 mice is pathogenic. 162 Another animal model of RP, the transgenic S334ter-3 rat, possesses the mutation RhoS334, which shows similar phenotypes to human class I RHO mistracking mutations, leading to a continual degeneration of photoreceptors and vision decline. 163 , 164 The protospacer adjacent motif (PAM) sequence in RhoS334 (5′-TGG-3′) diverges from the PAM in RhoWT (5′-TGC-3′) by only one nucleotide. Benjamin et al. reported that an allele-specific disruption of RhoS334 via a single subretinal injection of CRISPR/Cas9 and gRNA by electroporation prevented retinal degeneration and increased visual acuity. 165 Additionally, Latella et al. successfully edited the human rhodopsin (RHO) gene by the electroporation of plasmid-based CRISPR/Cas9 in a P23H transgenic mouse model for autosomal dominant RP and confirmed its efficacy as a genetic engineering tool in photoreceptor cells, 166 which strongly demonstrates that the CRISPR/Cas9 system is an efficient and promising therapeutic tool for retinal degeneration, such as RP. Suzuki et al. also determined a CRISPR/Cas9-mediated homology-independent targeted integration (HITI) strategy and demonstrated its efficacy in ameliorating visual function in a rat model of RP. 167 HITI is a targeted integration mediated by NHEJ, and this study is the first time that HITI could play a role in nonmitotic cells. The advantage of HITI technology is that it can be applied to any targeted genome engineering system, not just CRISPR/Cas9.

The combination of CRISPR/Cas9 technology and other methods provides new avenues for the treatment of related eye diseases, such as treatment with AAV and iPSCs. Bassuk et al. first reported that CRISPR/Cas9 precisely repairs retinitis pigmentosa GTPase regulator (RPGF) point mutations, which cause X-linked RP in patient-specific iPSCs; this supports that combining gene editing with autologous iPSCs could be a personalized iPSC transplantation strategy for therapies of various retinal degenerations. 168 Similarly, Deng et al. found that iPSC-derived retinal organoids from three RP patients with different frameshift mutations in the RPGR gene have significant defects in photoreceptors, including defects in their morphology, localization, and electrophysiological activity. The correction of an RPGR mutation via CRISPR/Cas9 reverses ciliopathy and rescues photoreceptor loss, which indicates that CRISPR/Cas9 can serve as an adopted mutation repair strategy. 169

LCA is a congenital retinal dystrophy that causes significant vision loss at an early age. 170 To verify that mutation in human KCNJ13 causes LCA, Zhong et al. employed CRISPR/Cas9 to create Kcnj13 mutant mice by zygote injection with sgRNA and spCas9 mRNA. Kcnj13 mutant mice showed a declined response to light, a loss of photoreceptors and rhodopsin mislocalization, revealing that the loss of Kcnj13 function could mimic human LCA phenotypes in mice. 171 As demonstrated by Zhong et al., CRISPR/Cas9 could accelerate the study of candidate gene function in biology and disease. 171 The centrosomal protein 290 kDa (CEP290) gene, the most frequent mutation in LCA, causes the most common subtype of LCA, which is referred to as LCA10. However, the large size of CEP290 exceeding the capacity of AAV delivery prevents the use of this delivery platform. To overcome this capacity limitation, Ruan et al. used dual recombinant AAV vectors to induce the CRISPR/Cas9-mediated deletion of a specific intronic fragment of the Cep290 gene in mouse photoreceptors. 172 Additionally, using a smaller S. aureus CRISPR/Cas9 system enables a single AAV vector to deliver the Cas9 gene and two gRNAs, which performs a dual-cut excision of the CEP290 mutation-containing region in primary fibroblasts from LCA10 patients. 164 Recently, Maeder et al. developed a candidate genome editing therapy named EDIT-101 to restore vision loss in LCA10. 173 They delivered the Staphylococcus aureus Cas9 and CEP290 gRNA to the photoreceptor via an AAV5 vector. Humanized CEP290 mice showed rapid and continuing CEP290 gene editing after subretinal delivery of EDIT-101. These extraordinary studies provide a roadmap for the preclinical advance of gene therapy for LCA10.

RB is the most common pediatric eye tumor of the developing retina. 174 Approximately one-third of RB cases are caused by biallelic RB1 mutation or deletion. Solin SL et al. reported that using TALEN gene editing to inactivate somatic rb1 in adult zebrafish induced tumorigenesis at high frequency. 175 A highly penetrant and rapid RB preclinical model was reported by Naert et al., utilizing the CRISPR/Cas9 system to induce the knockout of rb1 and retinoblastoma-like 1 (rbl1) in Xenopus tropicalis . 176 The animal model showed rapid development of RB, and it will be a good model for early stage drug discovery and rapid therapeutic target identification. Jian Tu et al. generated a pluripotent H1 human embryonic stem cell line with RB1 heterozygous knockout by CRISPR/Cas9 nickase, which provides a valuable cell resource for the study of hereditary retinoblastoma. 177 Glaucoma is the second leading cause of blindness worldwide and is characterized by elevated intraocular pressure (IOP). 178 Gain-of-function mutations in myocilin (MYOC) have been reported to commonly cause primary open-angle glaucoma (POAG). 179 , 180 , 181 The accumulation of mutated myocilin inside cells leads to the activation of the unfolded protein response (UPR) cascade and endoplasmic reticulum (ER) stress in the trabecular meshwork (TM). 182 , 183 TM cells are sensitive to chronic ER stress and finally die, resulting in increased IOP and glaucoma. 184 , 185 Jain et al. knocked down the expression of mutant MYOC in a mouse model of POAG by CRISPR/Cas9, resulting in the reduction of ER stress, lower IOP, and the preventability of further glaucomatous damage in mouse eyes. 186 Importantly, they also demonstrated the feasibility of utilizing CRISPR/Cas9 in human eyes with glaucoma. A dominant-negative mutation in KRT12, 187 which causes Meesmann epithelial corneal dystrophy (MECD), results in the occurrence of a novel Streptococcus pyogenes PAM. Courtney et al. designed a sgRNA complementary to the sequence adjacent to this PAM and found that this sgRNA has a large effect on the decrease in mRNA and protein of KRT12 in vitro. 188 The injection of combined Cas9/sgRNA into the corneal stroma of a humanized MECE mouse model showed frame-shifting deletions of the mutated KRT12 allele. This study is the first to demonstrate the in vivo allele-specific CRISPR/Cas9 gene editing of a novel PAM created by a heterozygous disease-causing SNP. 188

Hematological diseases

Nearly half of hemophilia A cases are caused by the inaccurate expression of factor VIII (F VIII) due to inversion of the chromosome. 189 In one study, iPSCs were derived from somatic cells of hemophilia A patients induced by chromosome inversion, and the F VIII gene of iPSCs was modified by CRISPR/Cas9 technology. 190 The modified iPSCs were induced to differentiate into mature endothelial cells capable of expressing factor VIII and then transplanted into hemophilia mice lacking factor VIII. The results showed that the transplanted mice began to produce factor VIII, which effectively inhibited bleeding symptoms. Hemophilia B is caused by a deficiency in factor IX (F IX). Coagulation activity can be restored by increasing FIX in plasma. Guan et al. 191 found that the F9 gene carries a new mutation, Y371D, in a family of hemophilia B patients, which leads to a more severe hemophilia B phenotype than the previously discovered Y371S mutation. They used naked DNA constructs and adenoviral vectors to deliver Cas9 to adult F9 Y371D mutant mice. After treatment, it was found that when adenovirus was used as a vector to deliver cas9, although the mutation gene was highly efficiently repaired, hepatotoxicity was severe. However, Cas9 with a naked DNA structure successfully repaired more than 0.56% of F9 alleles in hepatocytes in hemophilia B mice, enough to restore hemostasis. CRISPR technology also provides a quick path to build hemophilia models. Researchers from the Institute of Zoology in the Chinese Academy of Sciences injected the CRISPR/Cas9 system targeting vwF (vascular hemophilia mutant gene) into the fertilized eggs of miniature pigs and obtained the double allele mutant mini-pig quickly and efficiently. These miniature pigs have severe coagulopathy, indicating the successful construction of a miniature pig model of von Willebrand disease by CRISPR technology. 192

Sickle anemia is the first genetic disease with a clearly understood pathogenesis. A single nucleotide mutation from A to T in the first exon of human β-globin results in a lesion. 193 In 2016, a Stanford University team reported on the use of CRISPR/Cas9 technology to repair β-globin gene (HBB) mutations in patient-derived HSCs in vitro. 194 After the modified iPSCs differentiated into red blood cells, normal HBB mRNA could be detected. This preclinical experiment provided theoretical support for gene editing technology in the treatment of sickle anemia. The CRISPR/Cas9 system has also been used to correct β thalassemia-causing mutations in the HBB. 195 Using CRISPR/Cas9 technology to direct the calibrated DNA sequence to the HBB mutation site, it was possible to correct two different β-thalassemia mutations in the HBB gene of patient iPSCs by HR.

Other hereditary diseases

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations of the DMD gene. 196 Current X-linked muscular dystrophy (mdx) mice can only partially mimic human disease conditions. Their small size, limited chronic muscle damage and muscle weakness also impose limitations on disease research and analysis. Therefore, larger animals such as rats, rabbits or pigs are more valuable for preclinical studies. Larcher et al. 197 generated Dmd mdx rats by targeting exon 23 of DMD with TALENs. These edited rats showed a significant reduction in muscle strength and decreased spontaneous motor activity. Sui’s team generated DMD knockout rabbits by coinjecting Cas9 mRNA and sgRNA into rabbit zygotes targeting exon 51 of DMD. These rabbits harbored the typical phenotypes of DMD, and the pathological features in the diaphragm and heart were similar to those of DMD patients. 198 In addition, the monkey dystrophin gene was targeted using CRISPR/Cas9 to create mutations that cause DMD. The detection of the relative targeting rate showed that CRISPR/Cas9 could result in mosaic mutations in up to 87% of the dystrophin alleles in monkey muscle. 199 , 200 Notably, three groups of researchers have recently described the use of CRISPR/Cas9 to remove mutations in the DMD gene encoding dystrophin, which affects protein expression. 201 , 202 , 203 The investigators used the CRISPR/Cas9 system to excise the mutant portion of DMD in the mdx mouse model, thereby synthesizing a shorter version of dystrophin protein in the muscle fibers and restoring partial muscle function. This provided a promising method for correcting disease-causing mutations in the muscle tissue of patients.

Patients with primary immunodeficiencies lack a part of their immune system or have immune system dysfunction, and they can be treated with allogeneic HSC transplantation. 204 This may be a high-risk process when leukocyte antigen-matched donors lack tissue compatibility. Correcting a patient’s own HSCs through gene therapy provides an attractive option. HSCs can also be used in situ to correct pathogenic mutations and to develop cell or animal models to study the pathogenic effects of specific genetic defects found in immunodeficient patients. As the most severe immunodeficiency, severe combined immunodeficiency (SCID) is caused by a mutation in the gene encoding the interleukin 2 receptor gamma (IL2RG), which results in the developmental arrest of T cell production and additional primary or secondary defects in B cells. Several research teams have successfully used ZFN and TALEN techniques to induce HDR at the IL2RG locus in various human cell types, including HSCs and embryonic stem cells (ESCs). 205 , 206 , 207 Other studies have utilized endonucleases to generate different kinds of immunodeficient animal models that were previously unable to be established due to a lack of effective genetic modification. 207 , 208 , 209 , 210 As a result of engineered nuclease-mediated editing of genomic modifications, other animal disease models have been developed, simulating Rett syndrome, 211 hereditary deafness, 212 Wilson disease, 213 Laron syndrome, 214 Niemann–Pick disease, 215 Netherton syndrome, 216 and so on. Advances in genome editing technologies will further expand the application of animal models in disease mechanism research and treatment development.

Future application prospects

Genome editing in cancer immunotherapy.

Recently, cancer immunotherapy has stimulated great interest, with its goal to harness the patient’s own immune system against tumor cells. 217 One promising area in immunotherapy is the application of genetically engineered T cells, known as chimeric antigen receptor (CAR) T cells, which allow the targeting of tumor-associated antigens and could enhance the therapy response. 218 , 219 The preparation of functional CAR T cells requires several key steps (Fig. 3 ): first, the patient’s white blood cells are collected, and the patient’s T cells are isolated via leukapheresis, after which T cells are reengineered and modified with tumor-antigen-specific receptors and costimulating molecules; next, a CAR-containing viral vector is transduced into the modified T cells, followed by the amplification of the CAR-expressing T cells and then infusion of the cells into the patient. CARs are synthetic receptors that typically contain the following parts: an antibody-derived targeting ectodomain that recognizes tumor antigens; a costimulatory molecule region that can bind to receptors such as CD28, 4-1BB, or CD278; 220 and a T cell signaling domain. After binding to a particular antigen, the CAR can transmit signals and activate modified T cells. The independence of CAR recognition endows genetically engineered CAR T cells with a fundamental antitumor advantage by avoiding the limitation traditionally conferred by the major histocompatibility complex (MHC). 221 However, due to the complexity of the manufacturing process, the limited selection of target antigens and the insufficient antitumor responses to solid tumors, the applicability of this transformative product is highly limited. Over the past few years, flexible gene editing technologies have become significant engineering tools to address these limitations and further improve CAR T designs.

Production of CAR T cell products with genome editing technology.

The development of allogeneic CAR T cell therapy would simplify and solve some challenges in the process of manufacturing autologous CAR T cells. 222 The endogenous αβ T cell receptor (TCR) is responsible for major and minor histocompatibility antigen recognition. By genetically disrupting various parts of the αβ TCR complex and/or the human leukocyte antigen (HLA) class I loci of allogeneic T cells, it is possible to create a universal cellular therapy product that confers a wider range of application capability with minimal related adverse effects, including graft-versus-host disease (GVHD). In 2012, Torikai et al. used engineered ZFNs to eliminate the expression of α or β chains in endogenous TCRs, leading to the loss of TCR function in CD19 CAR T-cells. 223 These modified T cells did not respond to TCR-specific stimuli but retained the ability to recognize and target CD19, leading to the generation of universal allogeneic tumor-associated antigen-specific CAR T cells. With the same approach, the selective elimination of HLA expression was achieved in CD19-specific T cells and in embryonic stem cells, which increased the applicability of this strategy by avoiding the infusion of HLA-disparate immune cells. 224 Similar work was also performed by Poirot et al. using TALEN-mediated editing in 2015. By the application of TALEN-mediated gene editing, the expression of αβ TCR was inactivated, eliminating the possibility of T cell responses to allogeneic antigens and GVHD. 74 The beneficial role of TCR-depleted CD19 CAR T cells in evading GVHD has recently been validated in two infant patients with relapsed refractory CD19+ B cell acute lymphoblastic leukemia, leading to successful molecular remissions within 4 weeks. 225 In addition, the target of the lymphocytic depleting monoclonal antibody alemtuzumab, CD52, a human glycoprotein found on the surface of lymphocytes, was simultaneously disrupted by TALENs to eliminate the potential of any remaining alloreactive T cells and to promote the engraftment of cellular therapies. As a proof of application of this platform, TCR/CD52-deficient CAR T cells were administered concurrently with alemtuzumab and demonstrated antitumor activity in a lymphoma murine model similar to unmodified anti-CD19 CAR T cells, with resistance to alemtuzumab destruction. 226

The widespread use of gene editing techniques based on ZFNs and TALENs has been hampered by the requirement to design specific nuclease pairs for each new gene target. The development of the CRISPR/Cas9 system has successfully promoted multiple gene editing in CAR T cells in a faster and easier way. Using this technology, Liu et al. efficiently generated CAR T cells in which two (TRAC and B2M) or three genes (TRAC, B2M and PD-1) were simultaneously disrupted and tested their antitumor function in vitro and in vivo. 227 To target the first exon of TRAC and B2M, they designed four sgRNAs. To target the first exon of PD-1, two sgRNAs were designed, and one published sgRNA was tested. Finally, double-knockout (B2M and TRAC) T cells were induced with high efficiency, yet in triple-knockout (B2M, TRAC and PD-1) T cells, only 64.7% of the clones of the PD-1 PCR products were mutants, which implies that PD-1 expression might be downregulated during T cell expansion. More importantly, the CRISPR/Cas9-mediated multiplex gene-edited CAR T cells maintained CD19-specific antitumor function in a lymphoma xenograft mouse model, suggesting that they are promising reagents for cancer treatment. In another interesting study, 228 the efficient double knockout of endogenous TCR and HLA class I molecules was achieved by a one-shot CRISPR protocol that incorporated multiple gRNAs into a CAR lentiviral vector to generate allogeneic universal CAR T cells. In this study, CRISPR/Cas9 mediated the simultaneous knockout of four loci of the T cell surface receptors PD-1 and CTLA-4 and successfully generated allogeneic universal T cells. More recently, the CRISPR/Cas9-mediated generation of CAR T cells that specifically disrupt inhibitory immune receptors such as T cell membrane protein-3 (TIM-3), 229 adenosine 2a receptor (A2aR) 230 and lymphocyte-activation protein 3 (LAG-3) 231 have shown a better percentage of complete remission in xenograft mouse models by increasing the secretion of antitumor-related cytokines (such as IFN-g, GM-CSF and MIP-1b). These factors may be involved in CAR T cell exhaustion and acute myeloid leukemia (AML) dysfunction, as the combination of checkpoint inhibitors with CAR T cells may result in the enhanced antitumor efficacy of AML and other hematological malignancies.

Taken together, these results suggest that genome editing could serve as a good platform for generating “universal” CAR T cells and can be applied to the large-scale production of healthy “off-the-shelf” T cells against multiple targets.

Screening for functional genes

The concept of precision medicine has led to the development of many targeted drugs for the treatment of different diseases. For example, targeted drugs designed for known carcinogenic sites will specifically bind to carcinogenic components (gene fragment or protein) and induce the apoptosis of tumor cells without affecting normal tissue cells. However, one obvious drawback of this molecular targeting therapy is that a certain mutation or gene expression alteration is necessary for patients to respond to the targeted drug; otherwise, drug resistance persists. Based on CRISPR/Cas9 technology, scientists have established mammalian genome-wide mutation libraries or libraries of gene mutations associated with certain functions, which are related to screening phenotypes through functional screening and subsequent PCR amplification and deep sequencing analysis. The entire process is called the CRISPR/Cas9 gRNA library screening technology. 232 , 233 The gRNA library is an ideal tool for drug screening or the targeted screening of specific pathways. The establishment of gRNA libraries will play an important role in functional gene screening, disease mechanism research and drug development. Functional genome screening using the CRISPR system could reveal changes in gene expression after cancer drug therapy and help to investigate drug-gene interactions by adding small molecules as perturbations, thereby identifying novel targets for precise treatment and providing insights into disease development. 234 , 235

One of the chief goals of pooled CRISPR/Cas9 unbiased screening in cancer research is to identify genotype-specific vulnerabilities, and AML was the first disease to be systematically analyzed with this technology. 236 Using this platform, the authors found several well-known potential targets for AML therapies, including BCL2, BRD4, MEN1, and DOT1L, by studying five commonly used AML cell lines and two solid tumor cell lines as controls. Since then, large-scale CRISPR/Cas9 screening has been performed to systematically discover essential genes in many cancer cell lines 237 , 238 , and approximately 1500 essential genes have been identified, which is five times higher than the number of genes previously detected by shRNA screening. 239 Another successful example involved the use of CRISPR/Cas9-mediated loss-of-function screening to identify cancer metastasis-related genes. 240 In this study, a nonmetastatic lung cancer cell line was infected with the mouse genome-scale CRISPR knockout (mGeCKO) sgRNA library and subcutaneously transplanted into immunocompromised mice. After 6 weeks, enriched sgRNA sequencing was performed in mice with lung cancer metastasis, and several candidate genes related to lung metastasis were identified and verified, including the already known genes PTEN 241 , miR-345, 242 and miR-152 243 and several new genes, including Fga, Trim72 and Nf2. With a CRISPR-based strategy, another loss-of-function screening identified four candidate HCC suppressor genes that had not previously been associated with HCC (Nf1, Plxnb1, Flrt2, and B9d1). The authors also found that these suppressor genes were closely related to the RAS signaling pathway through the intervention of small molecule inhibitors. 244 A CRISPR-based double-knockout (CDKO) system has also been developed in K562 leukemia cells. The system uses dual sgRNA libraries to screen for combinatorial genes and identify pairs of synthetic lethal drug targets. 245 Recent landmark studies have demonstrated the power of CRISPR/Cas9 to discover long noncoding (lncRNA) loci. These studies applied CRISPR-interference (CRISPRi)- or CRISPR-activation (CRISPRa)-based libraries to screen for functional lncRNA loci that could modify cell proliferation 246 , 247 and drug resistance 235 , 248 . Generally, a comprehensive sgRNA library was designed to target the initiation site of lncRNA transcription, and then the library was transduced into different cell lines. Then, through sequence analysis, hundreds to thousands of lncRNAs promoting cell growth and drug resistance could be identified.

Depending on each mutation’s individual effect, the simultaneous mutation of two genes can produce an unexpected phenotype that determines the potential functional relationship between genes. 249 This phenomenon, known as genetic interaction, has implications for the development of cancer therapeutics; for example, in cancers with loss-of-function mutations in BRCA1 or BRCA2, an inhibitor of PARP1/2 (e.g., olaparib) could result in cell killing by simultaneously disrupting both genes. 250 The CRISPR/Cas9 system provides an effective strategy for identifying synergistic gene interactions to gain insights into the response of cancer to chemotherapy. A CRISPR-based double-knockout system combined with deep sequencing, phenotypic measurement and genetic analysis has identified interactions between the synergistic drug targets in K562 leukemia cells, such as BCL2L1 and MCL1. 245 Similarly, the double-knockout screening method was used to detect 73 tumor genes in pairs and found synthetic lethal interactions of many known (e.g., BRCA-PARP) and unknown genes, approximately 75% of which could be replicated using combinatorial drugs. 251 Combining pooled CRISPR screening with a perturbed drug could identify genes that synergize or confer resistance to the agent. 252 In one of the first pooled CRISPR screens, the BRAF inhibitor vemurafenib was used to treat a genome-scale knockout library of melanoma cells and recovered genes conferring resistance to the drug. 253 Similar to the CDKO system, another simple and effective strategy for analyzing the function of combinatorial genes is CombiGEM-CRISPR (combinatorial genetics en masse-CRISPR). 232 It combines two pooled sgRNA libraries in one vector, and some genetic hits (such as KDM6B and BRD4) were discovered by this method. Disrupting these genes with the CombiGEM system demonstrated a stronger synergistic effect on the proliferation of tumor cells compared to previously reported small molecule inhibitors. Likewise, a series of CRISPR-based screening techniques has been performed to identify genes that regulate cellular response to specific drugs, such as TRAIL, 254 ATR, 255 or Ras 256 pathway inhibitors. Of note, an in vivo screening based on CRISPR/Cas9 has identified protein tyrosine phosphatase nonreceptor type 2 (PTPN2) as a novel target for cancer immunotherapy. 257 In the future, this innovative approach could also be used to develop personalized cancer therapies based on genotype-specific targets. 258

Gene diagnostic tools

Cancer predisposition genes describe genes in which germline mutations result in an increased risk of cancer. 259 Identifying such sensitive genes through genetic diagnosis is critical for cancer prevention. However, low-frequency mutations are not easily identified by sequencing, and a CRISPR-based diagnostic system referred to as SHERLOCK (specific high sensitivity enzymatic reporter UnLOCKing) has been established to solve this problem. 260 Technically, the system consists of two important elements, the RNA-guided endonuclease Cas13a (another Cas family member) and the reporter signal. Cas13a exists as a key factor and effectively induces trans-cleavage of nonspecific single-stranded DNA (ssDNA). The reporter signal is released after RNA cleavage. This approach appeared to be a highly sensitive detection method when used to detect two cancer mutants, BRAF V600E and EGFR L858R. 57 Another system called DETECTR (DNA endonuclease-targeted CRISPR trans-reporter) has also been developed. 261 Cas12a acts like Cas13a in this system, and another enzyme, recombinase polymerase amplification (RPA), is used as a detection tool to screen for viral infections in cancer and to amplify microsamples. The system seemed to be a fast and inexpensive method for detecting HPV 16/18 in lung carcinomas. 262 In the study of breast cancer, the CRISPR nuclease-dead Cas9 (dCas9) system was fused to a DNA methyltransferase effector and infected healthy breast cells by lentivirus. Through this technology, researchers have discovered that the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene was a key driver in carcinogenesis, which led to abnormally rapid cell division and might become an early diagnostic marker for breast cancer. 263 Additionally, CRISPR/Cas9 gene editing as well as overexpression experiments have also confirmed that the BRCA1-delta11q optional splice isoform is a primary factor in PARPi and cisplatin treatment resistance in breast cancer. 264

Application of gene editing in clinical trials

Genome editing, as an attractive and challenging therapeutic approach, can correct or eliminate mutations that lead to the development of cancer and other genetically driven diseases. So far, ex vivo genome editing has been the most widely used, that is, the genetic engineering of cells in vitro and then the modified cells are re-engrafted back to patients. In recent years, teams represented by China and the United States have conducted a series of clinical trials of gene editing, such as producing more effective CAR T cells for the treatment of cancer and the knockout of the erythroid specific enhancer of BCL11A to upregulate gamma globulin in autologous erythroid HSCs as a potential therapy for sickle cell disease and β-thalassemia (Table 2 ).

Anticancer clinical trials

The gene editing clinical trial using the ZFN product GRm13Z40-2 for the treatment of stage III or IV malignant glioma patients (NCT01082926) was launched in 2010. The ZFN-mediated GRm13Z40-2, an allogeneic CD8+ cytolytic T cell line genetically modified to express the glucocorticoid-resistant IL13-zetakine, was delivered to tumor cells by intratumoral injection. In another phase I clinical trial (NCT02800369), ZFN agents (ZFN-603 and ZFN-758) were transfected into HPV-infected cervical epithelial cells to determine whether these agents could block the malignant progression of cervical intraepithelial neoplasia and reduce the incidence of cervical cancer. To date, this study has finished the data collection phase. Only two studies using TALENs in CAR T cells have been reported. One study (NCT02808442) developed a portfolio of allogeneic, universal CAR T cells (UCART19) that target relapsed or refractory CD19-positive B-acute lymphoblastic leukemia. In this study, alloreactivity and alemtuzumab sensitivity were eliminated by disrupting the loci encoding TRAC and CD52. A similar concept is used to generate allogeneic TALEN-edited CAR T cells that target CD123 (UCART123) in AMLs and blastic plasmacytoid dendritic cell neoplasms (NCT03190278).

Due to the simple design process and the ability to make multiple gene edits at one time, the CRISPR/Cas9 system has become an important tool in the development of cancer therapy. To date, eleven clinical trials have been carried out to assess the effectiveness of the CRISPR system in cancer therapy, seven of which are immunotherapies that target PD-1 protein expression. The first clinical trial using the revolutionary CRISPR/Cas9 technique for cancer treatment recruited its first patient in West China Hospital, Sichuan University in 2016. 265 In this nonrandomized, open-label phase I study (NCT02793856), the safety of ex vivo engineered PD-1 knockout T cells has been evaluated in the treatment for metastatic non-small cell lung cancer with progression after all standard treatments. In this trial, PD-1 expression was disabled by CRISPR/Cas9 in peripheral blood lymphocytes harvested from the enrolled patients. The edited lymphocytes were isolated, expanded and subsequently reinfused into the patients. Ongoing clinical trials apply the same concept of PD-1 knockout autologous T cells to treat other cancer types, including prostate cancer (NCT02867345), esophageal cancer (NCT03081715) and renal cell cancer (NCT02867332). These trials can be considered as the first proof-of-concept studies to apply the in vitro CRISPR/Cas9 gene knockout technique in cancer therapy. There are now studies combining PD-1 knockout with other targeted editing in therapy development, which may lead to improved efficacy for clinical application. One example is the addition of PD-1 knockout to Epstein–Barr virus (EBV)-specific autologous T cells for the treatment of EBV-positive cancers, which is currently in phase I/II clinical trials (NCT03044743).

The elimination of endogenous TCR and PD-1 by CRISPR might enhance tumor rejection activity. Recently, the Recombinant DNA Advisory Committee (RAC) of the US National Institutes of Health (NIH) approved a clinical trial to be piloted at the University of Pennsylvania. In this trial, PD-1 and the endogenous TCR will be abolished by CRISPR/Cas9 in HLA-A*0201 restricted NY-ESO-1 TCR redirected autologous T cells. Such redirected engineered T cells will be applied to a variety of cancer types, including relapsed refractory multiple myeloma, melanoma, synovial sarcoma, and myxoid/round cell liposarcoma (NCT03399448).

The use of CRISPR/Cas9 technology to generate CAR T cells to attack malignant cells has become a research hotspot in clinical trials. A clinical phase I/II trial (NCT 03166878) was conducted to evaluate the safety and tolerance of patients with recurrent or refractory CD19+ leukemia and lymphoma to several doses of universal CD19-specific CAR T cells (UCART 019). In this study, UCART019 cells were obtained by combining lentiviral delivery of CAR receptors and CRISPR RNA electroporation to simultaneously disrupt endogenous TCR and B2M genes. These cells are derived from one or more healthy unrelated donors but might help to avoid graft-versus-host-disease (GVHD) and reduce host-mediated immunity, thereby providing patients with anti-leukemic effects in a relatively safe condition. Unfortunately, a small number of patients relapsed due to the lack of CD19 expression in tumor cells. Therefore, another clinical trial (NCT03398967) that is more applicable for a wide range of patients focused on allogenic CRISPR-edited bispecific CD19+CD20+ or CD19+CD22+ CAR T cells, which could recognize and kill the CD19-negative malignant cells through the recognition of CD20 or CD22. In another study, a new clinical trial (NCT03057912) has proposed to evaluate the safety and efficacy of combination genome editing of TALENs and CRISPR/Cas9 by targeting HPV16 and HPV18 E6/E7 DNA in the treatment of HPV-associated cervical intraepithelial neoplasia. In this trial, CAR T cells edited by both techniques were administered twice a week for 4 weeks to disrupt target gene expression and promised to reduce off-target effects.

The mutation rate of the neurofibromatosis type 1 (NF1) gene is one of the highest in the human genome, which is likely to cause various benign or malignant tumors. 266 In one trial (NCT03332030), CRISPR/Cas9 technology was designed to screen and identify NF1-specific drugs. First, a human iPSC library was established from NF1 patients with good phenotypic characteristics, and different cell lines (NF1+/+, NF1+/− and NF1−/−) were developed using CRISPR/Cas9. Then, potential therapeutic agents could be identified by examining the reversal or remission phenotypes after specific drug use. Although results from clinical trials in genome editing appear to be promising, more work needs to be done to ensure the safety and effectiveness of this tool in treating human cancers.

Antiviral clinical trials

CCR5 acts as a major coreceptor in the early stage of HIV infection, and CXCR4 plays an important role as an auxiliary receptor when establishing stable infections. Treatment strategies targeting both coreceptors may avoid protection failure because coreceptor usage of HIV infection can be switched between CCR5 and CXCR4. 267 The production of engineered immune cells resistant to HIV infection or replication is the primary strategy for genome editing-based HIV treatment. The most common method involves two steps: modifying the cells (CD4+ T cells and CD34+ hematopoietic stem/progenitor cells) in vitro and then reinfusing the modified cells into patients. 268 , 269 Several clinical trials involving CD4+ T cell modification in the context of HIV infection have already been tested. The first approved genome editing trial involving the treatment of HIV with ZFNs (NCT00842634) began in 2009 to evaluate the safety and anti-HIV effects of modified autologous CD4+ T cells in HIV-1 infected patients. The ZFNs were delivered ex vivo to autologous CD4+ T cells by adenoviral vectors for CCR5 gene knockout, and each participant received a single infusion of 5–10 billion ZFN-modified CD4+ T cells. The clinical outcome was published in 2014 270 and indicated that CCR5-knockout cells were protected from CCR5-tropic HIV infection, and the infusions of genetically engineered T cells into patients were well tolerated, with only 1 patient presenting with minor infusion-related adverse events. Since the preliminary demonstration of clinical safety, the main purpose of follow-up trials has been to further optimize the therapeutic effect of gene-edited T cells. Sangamo Therapeutics Inc. and the University of Pennsylvania tried to improve engraftment of the infused T cells by increasing the number of genetically modified CD4+ T cells, clearing nonmyeloablative lymphocytes, using multiple infusions of cells and switching from adenoviral vector delivery to mRNA electroporation. Although recent advances in ZFN-modified CD4+ T cell infusion have provided some evidence for the safety and low off-target rate of this therapy, a long-term evaluation is still needed. Another study provided proof for the safety of the permanent gene disruption of CCR5 in autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) with ZFN ex vivo (NCT02500849). The main advantage of using HPSCs over T cells is that we will be able to obtain a large number of cell subsets that are protected from HIV infection, which are differentiated by the genetically edited CD34+ population. A recently reported article showed that Chinese scientists have established a CRISPR/Cas9-modified CCR5 gene editing system for adult HPSCs to achieve long-term and stable hematopoietic system reconstruction after infusion of modified CD34+ cells into patients with HIV-1 infection and ALL (NCT03164135). 271 This study preliminarily proved the feasibility and safety of gene editing adult HPSC transplantation in the human body and would promote the development of gene editing technology in clinical applications. Because HSPC-based gene therapy is often confined by ex vivo culture techniques and difficulties in HPSC expansion, there is also interest in modifying patient-specific iPSCs and reprogramming them to HSPCs. 272 (Clinical trials involving HPV and EBV infection are described in the Anticancer clinical trial section).

Clinical trials of hematological diseases

To date, ZFN and CRISPR/Cas9 have been applied in five clinical gene-therapeutic trials pertaining to hematological diseases, including hemophilia B, β-thalassemia, and sickle cell disease.

Hemophilia B is a recessive, X-linked hemorrhagic disease represented by a lack of expression of coagulation factor IX (F IX). 273 In November 2016, Sangamo Therapeutics Inc. initiated a phase I clinical trial (NCT02695160) with the expected 12 participants using SB-FIX, which is an AAV-delivered ZFN, designed to be intravenously delivered to the subject’s own hepatocytes to insert a corrective FIX transgene into the albumin locus; thus, they aim to achieve permanent FIX clotting factor production in the liver of severe hemophilia B patients. This ascending dose phase I study attempts to assess the safety and tolerability of SB-FIX in treating hemophilia B patients and is expected to be complete in January 2021. Abnormality in the β-globin gene (HBB) can reduce the synthesis of β-globin chains in hemoglobin, causing β-thalassemia. 274 In January 2019, Allife Medical Science and Technology Co., Ltd. started a 12-subject early phase I trial, where they applied CRISPR/Cas9 to correct the HBB gene in vitro in patient-specific induced hematopoietic stem cells (iHSCs), and intravenously transfused the edited cells back to the HBB-mutated β-thalassemia subjects. This trial is expected to be complete in 2021. BCL11A, a key modifier in hemoglobin disorders characterized by repressing fetal hemoglobin (HbF), is associated with the clinical severity of β-thalassemia and sickle cell disease. 275 Hence, gene therapy targeting BCL11A to treat the two diseases above has been tested in trials. Until now, three trials have tried to suppress the BCL11A gene in autologous CD34+ hematopoietic stem/progenitor cells in vitro and then intravenously transfuse the modified cells back to the subjects; all three trials initiated in 2018 and are expected to be complete in 2020–2022. Sangamo Therapeutics Inc. has led the first trial, NCT03432364, a single-dose phase I/II study with 6 subjects of transfusion-dependent β-thalassemia (TDT). ZFN has been applied to generate the gene-edited therapeutic cell ST-400; its safety, tolerability, and effects on HbF are to be evaluated and its transfusion requirements are to be assessed. Another single-dose phase I/II study trial (NCT03655678) with up to 45 subjects, focusing on transfusion-dependent β-thalassemia (TDT), was initiated by Vertex Pharmaceuticals Inc. They utilized CRISPR/Cas9-modified cell CTX001, aiming to test its safety and efficacy. With similar study designs and start and completion times, Vertex Pharmaceuticals Inc. have also tested CTX001 in severe sickle cell disease (NCT03745287).

Clinical trials of hereditary eye diseases

Gene augmentation is successfully employed for the treatment of inherited retinal diseases, and a large number of clinical trials of gene augmentation are underway for LCA, choroideremia, achromatopsia, X-linked retinoschisis and RP. 276 Until now, there has been only one clinical trial of gene editing in LCA10. Recently, a clinical study (NCT03872479) was initiated by Editas and Allergan to evaluate the safety, tolerability and efficacy of single-dose AGN-151587 (EDIT-101), an AAV vector containing 3 components: an S. aureus Cas9 and two gRNAs –gRNA-323 and gRNA-64. AGN-151587 could eliminate the mutation of c.2991 + 1655A > G in intron 26 of the CEP290 gene to treat LCA10. Although clinical trials on gene editing for ophthalmic diseases have just begun, the unique qualities of eyes, such as easy accessibility and relative immune-privileged status, make CRISPR–Cas a promising and available strategy for ophthalmic disease treatment in the near future. 164 , 276

Challenges in therapeutic targeting

In addition to the many benefits of genome editing, there are some technical challenges in translating these treatments to clinical disease therapy, primarily in terms of accuracy, efficacy and delivery hurdles. To cope with these challenges, scientists will need profound knowledge about the molecular nature of cancers, especially heterogeneous solid tumors, as well as carefully designed genome editing platforms in preclinical studies.

Increasing the specificity of gene correction

The accuracy of gene editing technology is defined by the ability to edit the desired locus of interest within the genome. Mutations in undesired genomic loci, namely off-target effects, are inevitably rather pernicious, as they can lead to potential genomic toxicity, genome instability, the disruption of gene function, epigenetic alterations, and even carcinogenesis. 16 , 277 , 278 Given that therapeutic gene targeting is strongly dependent on the creation of DSBs at specific target sites, assays of paramount importance have been developed to assess the targeting specificities of ZFNs, TALENs and Cas9 nucleases, such as in vitro selection libraries, 279 , 280 mismatch-detection nuclease assay, 281 newly reported high-throughput profiling, 282 next-generation sequencing (NGS) 283 and whole-genome sequencing (WGS). 284 , 285 Thus, the above studies revealed a number of factors that might affect the specificity of gene editing, which can be roughly divided into two categories. First, the intrinsic specificity encoded in the Cas9 protein may determine the relative importance of each position that may differ between different sgRNA sequences. Second, the specificity also depends on the abundance of effective nuclease complexes relative to the target concentration.

Compared to ZFNs and TALENs, CRISPR/Cas9 may present higher potential for off-target effects in human cells. 278 As previously noted, there is a tolerance of sequence mismatch when Cas9-sgRNA binds to the target DNA: both identical and highly homologous DNA sequences can be cleaved, leading to chromosomal rearrangements or off-target mutations. 286 , 287 With numerous studies demonstrating the presence of its off-target activity, it has become the task with top priority to improve DNA specificity in CRISPR technology. 278 Accordingly, several strategies have been exploited to minimize Cas9-mediated off-target effects and increase the cleavage specificity. Both the structure and composition of gRNA can affect the level of off‐target effects. 288 , 289 A related method that has been reported to reduce the off-target effects induced by Cas9 is to choose unique target sequences that lack homology to other regions of the genome. 290 In addition, the use of truncated and less-active sgRNAs that are shortened at the 5ʹ end by two to three nucleotides decreased undesired mutagenesis at some off-target sites because this sgRNA structure has higher sensitivity to mismatches. 277 , 282 Another strategy to reduce the off-target effects is to harness a pair of nCas9 or RNA-guided FokI nucleases to generate paired nicks instead of DSBs, which can significantly avoid off-target cleavage without sacrificing genome editing efficiency. 291 , 292 In addition, the concentration of Cas9-sgRNA delivered to cells should be carefully controlled, as it is another factor that affects off‐target effects. 293 However, increasing specificity by reducing the amount of transfected DNA also results in reduced cleavage at the target. Therefore, a balance between on-target cleavage efficiency and off-target effects must be considered. Most recently, two different variants of monomeric Streptococcus pyogenes have been engineered to form a SpCas9 that exhibits improved genome-wide specificities. Slaymaker et al. described an enhanced SpCas9 that contains alanine substitutions at three positions and predicted the interaction of this variant with a nontarget DNA strand. 294 In another study, Kleinstiver et al. created SpCas9-HF1 (high-fidelity variant 1) by introducing alanine substitutions at four residues in SpCas9 to disrupt nonspecific contacts with the phosphorylated framework of the target DNA strand, which interacts with gRNA. 295 These engineered variants of SpCas9 have been engineered by reducing nonspecific interactions of proteins with different DNA strands, dramatically improving genome-wide specificity. They do not alter the target range or size of the DNA that is required to encode the desired Cas9 nuclease and a single gRNA; thus, functional mutations could also be combined to further increase specificity.

Alternative delivery methods have also been developed to improve the specificity of the editing process. Direct delivery of recombinant Cas9 protein and in vitro transcribed sgRNA either alone or in purified complexes reduced off-target effects when compared with plasmid transfected delivery systems. 296 , 297 Anti-CRISPR molecules, recently discovered inhibitors for CRISPR systems, may add the precise control of genome editing strategy, 298 which are currently tested. 299

Improving the efficiency of nuclease editing

The efficiency of DSB repair pathways mediated by NHEJ and HDR varies greatly between cell types and cell status; however, in most cases, NHEJ is more active than HDR. It has been observed that NHEJ is active throughout the cell cycle of a variety of cell types, including division and postmitosis. 11 , 300 In contrast, HDR functions primarily in the S/G2 phase and is therefore largely restricted to actively dividing cells, limiting treatments for the precise genomic modification of mitotic cells. 301 , 302 This difference makes the treatment of diseases that require genetic correction or gene insertion more challenging than those that require gene inactivation. Since NHEJ-mediated DSB repair can be applied to promote high levels of gene disruption in most cell types, the primary challenge to date has been to improve the efficiency of HDR.

Notably, recent studies have reported novel strategies to upregulate the efficiency of genome editing by inhibiting competing DNA repair pathways, primarily NHEJ-mediated DNA repair. Maruyama et al. 303 successfully employed SCR7 to inhibit NHEJ by targeting a key enzyme (DNA ligase IV) in the NHEJ pathway, thereby increasing the genome editing efficiency in cell lines and mice by up to 19-fold. In another independent study, Kuhn et al. abolished NHEJ activity in human and mouse cell lines by the gene silencing of several key molecules of the NHEJ repair pathway (KU70, KU80 or DNA ligase IV), leading to increased genome editing efficiency. 304 Further, Canny et al. discovered that 53BP1, a genetically encoded inhibitor, increased HDR-dependent genome editing efficiency by up to 5.6-fold through suppressing NHEJ activity in human and mouse cells. 305 Interestingly, by application of an HDR enhancer, RS-1, Song et al. achieved multifold improvement on the CRISPR/Cas9- and TALEN-mediated knock-in efficiency both in vitro and in vivo, whereas the NHEJ inhibitor SCR7 has minimal effects. 306 The identification of novel small molecule inhibitors against other NHEJ proteins, such as artemis and XRCC4, may further advance current strategies. 307 , 308 An improved CRISPR system, called CRISPR/Cpf1 or CRISPR/Cas12a, that employs a smaller and simpler RNA-guided DNA nuclease, could target genomic regions that cannot be targeted by Cas9 and induce multiplex gene perturbation in vitro with frequencies of up to 45%. 309 In addition, timed delivery of Cas9-guide RNP (RNA ribonucleoprotein) complexes was used to site-specifically induce DSBs and new genetic information, with high efficiency of HDR. 310 In addition to the methods already mentioned, further research aimed at improving HDR efficiency will be necessary to optimize genome editing for a wider range of diseases.

Although the CRISPR/Cas9 gene editing system improves the efficiency of gene knockout and site-directed modification (including site-directed mutation and gene insertion), the efficiency of gene site-directed mutation based on a HR mechanism is still low. To improve the efficiency of site-directed mutation, the base editor (BE) system combining CRISPR/Cas9 and cytosine deaminase has been reported one after another. 311 , 312 , 313 By using this system, the fusion protein composed of Cas9-cytidine deaminase and uracil glycosaminase inhibitor can be targeted at the desired site complementary to gRNA without double-stranded DNA fragmentation, and the amino group of pyrimidine (C) at the target site can be removed so that C becomes uracil (U), and U will be replaced by thymidine (T) with the replication of DNA. Finally, the single base C → T mutation is realized accurately and efficiently, leading to single-base-pair substitutions in eukaryotic cells. 314 The BE technique adds an important tool to the research and application of genome editing technology.

Optimizing the delivery system

One of the key challenges for the future application of gene editing tools will be the development of efficient and secure methods to deliver genetic editing elements, not only to the tumor cells ex vivo but also to somatic cells in vivo. Delivery methods include viral methods and nonviral physical methods (Fig. 4 ). Nonviral physical delivery methods, such as electroporation, 315 hydrodynamic injection 316 and lipid nanoparticles, 317 have been widely utilized to deliver ZFNs, TALENs, and CRISPR in different cell lines and animal models. Despite their simplicity and safety, the relatively poor delivery efficacy limits the therapeutic applications of those nonviral delivery methods in vivo. 318 In contrast, viral vectors (such as retroviruses, lentivirus, adenovirus (AdV) and AAV) have high delivery efficiency, and some of them have been approved for clinical uses. 319 , 320 To date, viral delivery systems have been the most effective system for delivering plasmid-based nucleic acids to mammalian cells in vitro and in vivo, despite the possibility of introducing unintentional mutations and the existence of safety concerns. 321 , 322 , 323 , 324 Recent studies have further highlighted other issues affecting delivery efficiency, including the immune risk of host tumors and cells to Cas9 proteins, 325 as well as the DSB P53 responses related to genome editing. 326 Many new viral and nonviral systems have been developed to overcome these problems.

Viral and nonviral delivery systems for genome editing technology. The most commonly used viral vectors include adeno-associated viruses (AAVs), lentiviruses and adenoviruses (AdVs). Nonviral physical methods can be used for genome editing to deliver biomacromolecules intracellularly without the use of nanoparticles. Nonviral delivery may be microinjections in vitro, direct injection into the embryo or zygote ex vivo, or hydrodynamic injection in vivo. Alternatively, electroporation or mechanical deformation realize delivery by creating transient pores in the cellular membrane, making entry points for genome editing biomacromolecules.

Nonviral delivery systems could extend the range of genome editing therapies by alleviating concerns about the safety and immunogenicity of native cells in vivo. For instance, the delivery of plasmid DNA encoding a Cas9-sgRNA complex that targets VEGF using a PEG-PEI-cholesterol lipid polymer could achieve a gene knockout of approximately 50% in osteosarcoma cells in vitro and in vivo. 327 A lipid delivery system containing PEG-poly lactic-coglycolic acid nanoparticles was used to deliver CRISPR DNA constructed by a CD68 promoter and achieve in vitro and in vivo gene editing of specific macrophages. 328 Zuris and colleagues also studied lipid materials as vectors for genome-edited proteins. First, they fused Cas9 and TALEN into anionic GFP proteins to increase negative charges on the surface and then complexed them with Lipofectamine 2000TM (a commercially cationic lipofection reagent); this novel complex achieved 24% gene knockout of mouse embryonic stem cells in vitro and 13% gene knockout of mouse cochlea hair cells in vivo. 317 The gene knockout rate of the complex to mouse embryonic stem cells in vitro was 24%.

According to Finn et al., lipid nanoparticles composed of PEG–lipids exhibited excellent serum stability. When used to deliver Cas9 mRNA and sgRNA targeting the mouse transthyretin gene in hepatocytes, they caused a drop in serum protein levels of more than 97%, which lasted for at least 12 months after a single systemic injection. 329 Recent work by Cheng and Leong et al. has demonstrated that the delivery of Cas9 and sgRNA plasmids with cationic alpha-helical polypeptides is expected to enhance gene editing efficiency in vitro and in vivo. With this delivery system, repeated intratumoral injections in a HeLa xenograft mouse model resulted in ~67% targeted gene knockdown and >71% tumor growth inhibition and ultimately significantly prolonged the survival of tumor-bearing mice. 330 Moreover, the Cas9 protein and sgRNA complex showed higher efficiency than plasmid-based CRISPR/Cas9 and Cas9 mRNA/sgRNA. For example, recombinant Cas9 proteins and sgRNA have been reported to achieve 16% editing efficiency in vitro through cell-penetrating peptide (CPP), 297 , 331 while the delivery of purified Cas9 protein mediated by electroporation increased the editing efficiency to 79%, 296 , 332 because transgenic proteins degraded rapidly and avoided long-lasting effects on the genome.

To improve the specificity and safety of viral-mediated gene editing delivery, different parts of preexisting viruses can be mixed together, creating hybrid virus vectors. The structure of the virus can be tweaked by point mutations, or the virus can incorporate small molecules, synthetic polymers and inorganic nanoparticles. 333 For example, lentiviral vectors are typically pseudotyped with glycoprotein G from vesicular stomatitis virus (VSV-G), extending the vector tropism to a wide range of host cells. 334 By controlling the ratio of assembled wild-type viral capsid to protease-activatable subunits, the overall transduction level of protease-activatable viruses (PAVs) increased. 335 Using error-prone polymerase chain reaction (EP-PCR), Asuri et al. created a library of AAV capsid genes with point mutations, which resulted in a viral variant that was more efficient in delivering genetic payloads to human stem cells. 336 The vector is further enhanced by conjugative delivery to ZFNs: the induced DSB facilitated HDR repair of the delivered transgene, thereby enabling gene targeting. Another way to further modify or enhance the functional properties of viruses is by incorporating synthetic nonbiological components such as polymers and nanoparticles. Hofherr et al. 337 attached PEG-5000 to adenoviral vectors to generate adeno-PEG-injected counterparts (Ad-PEG). After intravenous injection into mammalian blood, PEGylation blunted the interactions of adenovirus with platelets and endothelial cells and reduced thrombocytopenia as well as D-dimer formation. In another study, Lee et al. investigated the possibility of conjugating the AAV surface-exposed lysine on the capsid with the activated PEG chains of PEG-2000 to protect the AAV vectors from neutralizing antibodies. 338 At a critical conjugation ratio, the particles were moderately protected from serum neutralization by 2.3-fold over the unmodified vectors. These results indicate that certain modifications of viral vectors may have utility to reduce immune responses that are involved in the delivery process, thereby improving their safety for human gene therapy.

The proper selection of different delivery systems and CRISPR/Cas9 types also contributes to the reduction of off-target effects. For instance, the use of minicircle DNA is more efficient and less immunogenic than plasmid DNA per-mass due to the elimination of bacterial expression sequences. 339 The codelivery of Cas9 and EGFR mutation-specific sgRNAs by adenovirus could precisely disrupt the oncogenic mutant allele, showing high specificity. 340 Furthermore, nonviral polymers conjugated to the gold nanoparticle hybridization system have been recognized as a suitable vehicle for the delivery of Cas9 RNP complexes plus donor DNA, which could effectively correct the disease phenotypes of muscle cells after intramuscular injection. 341

Conclusions and future perspectives

The research evidence accumulated to date has demonstrated significant contributions of genome editing systems to exploit therapeutic strategies for various types of human diseases, among which the CRISPR/Cas9 system has been especially effective by directly interfering with target gene loci or deriving multifunctional tools. In the future, a combination of pooled CRISPR screening and the existing information on the genetic and epigenetic characteristics of cancer cell lines will be able to broadly identify synthetic lethal interactions in the genome and facilitate the discovery of novel drug targets. The CRISPR/Cas9 platform also provides a new tool to manipulate noncoding regions of the cancer genome, accelerating the functional exploration of aspects that are hitherto poorly characterized. The tremendous advances in the development of engineered nucleases (especially ZFNs, TALENs, and CRISPR/Cas9) paved the way for genome editing from a theoretical concept into clinical practice. At the end of 2017, Brian Madeux, an American man with Hunter’s syndrome, received a bold treatment at the Benioff Children’s Hospital at the University of California–San Francisco—the delivery of ZFNs via an AAV for in vivo genetic editing to treat his disease. This is the first report in the world describing the treatment of genetic diseases through in vivo gene editing, which further demonstrates that gene editing has extremely important clinical application potential for the treatment of genetic diseases. Genome editing technology has also been combined with tumor immunotherapy to provide more updated options for human disease treatment. As one of the most innovative and successful approaches in tumor immunotherapy, CAR T cell therapy was officially approved for use in the clinic in 2017. Refractory ALL and CLL patients responded completely to CAR T cell products directly targeting CD19, therefore the US Food and Drug Administration (FDA) recognized CAR T cell therapy as a “breakthrough therapy” and approved its treatment for leukemia and lymphoma. The effective response of CAR T therapy in clinical trials of B cell malignancies has evoked great enthusiasm for the ultimate intelligent treatment, brought hope to cancer patients, and led to the commercialization of CAR T cells by many pharmaceutical and biotechnology companies. However, the development of CAR T cell therapy is still in its infancy, and the high costs of CAR T cell therapy have made it unaffordable for a large population in society. Moreover, the commercial potential of this therapy, especially the possibility of becoming an off-the-shelf therapy, remains uncertain; in addition, its capacity to combat solid tumors remains to be confirmed.

At the same time, gene editing technology has also promoted the development of cell imaging, gene expression regulation, epigenetic modification, therapeutic drug development, functional gene screening, and gene diagnosis. Although the off-target effect in the implementation of gene editing technology still needs further optimization, innovative genome editing complexes and more specific nanostructured vehicles have improved efficiency and reduced toxicity during the delivery process, bringing genome editing technology closer to the clinic. With deeper exploration into this technology and the cooperation of the world scientific community, it is reasonable to believe that genome editing technology has the potential to ultimately elucidate biological mechanisms behind disease development and progression, thus providing novel therapies and finally promoting the development of the life sciences.

Rothstein, R. J. One-step gene disruption in yeast. Meth. Enzymol. 101 , 202–211 (1983).

Article   CAS   Google Scholar  

Cornu, T. I., Mussolino, C. & Cathomen, T. Refining strategies to translate genome editing to the clinic. Nat. Med. 23 , 415–423 (2017).

Ghosh, D., Venkataramani, P., Nandi, S. & Bhattacharjee, S. CRISPR-Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics. Cancer Cell Int. 19 , 12 (2019).

Article   PubMed   PubMed Central   Google Scholar  

Gaj, T., Gersbach, C. A. & Barbas, C. F. 3rd ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31 , 397–405 (2013).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Rouet, P., Smih, F. & Jasin, M. Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc. Natl Acad. Sci. USA 91 , 6064–6068 (1994).

Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36 , 765–771 (2018).

O’Driscoll, M. & Jeggo, P. A. The role of double-strand break repair—insights from human genetics. Nat. Rev. Genet. 7 , 45–54 (2006).

Article   PubMed   CAS   Google Scholar  

Kaniecki, K., De Tullio, L. & Greene, E. C. A change of view: homologous recombination at single-molecule resolution. Nat. Rev. Genet . https://doi.org/10.1038/nrg.2017.92 (2017).

Article   PubMed   PubMed Central   CAS   Google Scholar  

Kim, H. & Kim, J. S. A guide to genome engineering with programmable nucleases. Nat. Rev. Genet. 15 , 321–334 (2014).

Article   CAS   PubMed   Google Scholar  

Verma, P. & Greenberg, R. A. Noncanonical views of homology-directed DNA repair. Genes Dev. 30 , 1138–1154 (2016).

Chang, H. H. Y., Pannunzio, N. R., Adachi, N. & Lieber, M. R. Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat. Rev. Mol. Cell Biol . 19 , 191–207 (2017).

Lieber, M. R., Gu, J., Lu, H., Shimazaki, N. & Tsai, A. G. Nonhomologous DNA end joining (NHEJ) and chromosomal translocations in humans. Subcell. Biochem. 50 , 279–296 (2010).

Delacôte, F. & Lopez, B. S. Importance of the cell cycle phase for the choice of the appropriate DSB repair pathway, for genome stability maintenance: the trans-S double-strand break repair model. Cell Cycle 7 , 33–38 (2008).

Article   PubMed   Google Scholar  

Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet 11 , 636–646 (2010).

Silva, G. et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr. Gene Ther. 11 , 11–27 (2011).

Cathomen, T. & Keith Joung, J. Zinc-finger nucleases: the next generation emerges. Mol. Ther. 16 , 1200–1207 (2008).

Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326 , 1509–1512 (2009).

Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29 , 149–153 (2011).

Al-Attar, S., Westra, E. R., van der Oost, J. & Brouns, S. J. Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes. Biol. Chem. 392 , 277–289 (2011).

Cox, D. B., Platt, R. J. & Zhang, F. Therapeutic genome editing: prospects and challenges. Nat. Med. 21 , 121–131 (2015).

Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339 , 819–823 (2013).

Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339 , 823–826 (2013).

Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93 , 1156–1160 (1996).

Diakun, G. P., Fairall, L. & Klug, A. EXAFS study of the zinc-binding sites in the protein transcription factor IIIA. Nature 324 , 698–699 (1986).

Beerli, R. R. & Barbas, C. F. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 20 , 135–141 (2002).

Beerli, R. R., Schopfer, U., Dreier, B. & Barbas, C. F. Chemically regulated zinc finger transcription factors. J. Biol. Chem. 275 , 32617–32627 (2000).

Buck-Koehntop, B. A. et al. Molecular basis for recognition of methylated and specific DNA sequences by the zinc finger protein Kaiso. Proc. Natl Acad. Sci. USA 109 , 15229–15234 (2012).

Fairall, L., Schwabe, J. W., Chapman, L., Finch, J. T. & Rhodes, D. The crystal structure of a two zinc-finger peptide reveals an extension to the rules for zinc-finger/DNA recognition. Nature 366 , 483–487 (1993).

Guo, J., Gaj, T. & Barbas, C. F. Directed evolution of an enhanced and highly efficient FokI cleavage domain for zinc finger nucleases. J. Mol. Biol. 400 , 96–107 (2010).

Smith, J. et al. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res. 28 , 3361–3369 (2000).

Beumer, K. J. et al. Efficient gene targeting in Drosophila by direct embryo injection with zinc-finger nucleases. Proc. Natl Acad. Sci. USA 105 , 19821–19826 (2008).

Paschon, D. E. et al. Diversifying the structure of zinc finger nucleases for high-precision genome editing. Nat. Commun. 10 , 1133 (2019).

Bogdanove, A. J., Schornack, S. & Lahaye, T. TAL effectors: finding plant genes for disease and defense. Curr. Opin. Plant Biol. 13 , 394–401 (2010).

Bogdanove, A. J. & Voytas, D. F. TAL effectors: customizable proteins for DNA targeting. Science 333 , 1843–1846 (2011).

Moscou, M. J. & Bogdanove, A. J. A simple cipher governs DNA recognition by TAL effectors. Science 326 , 1501 (2009).

Li, T. et al. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucleic Acids Res. 39 , 359–372 (2011).

Mussolino, C. et al. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 39 , 9283–9293 (2011).

Mercer, A. C., Gaj, T., Fuller, R. P. & Barbas, C. F. Chimeric TALE recombinases with programmable DNA sequence specificity. Nucleic Acids Res. 40 , 11163–11172 (2012).

Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39 , e82 (2011).

Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30 , 460–465 (2012).

Briggs, A. W. et al. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Res. 40 , e117 (2012).

Schmid-Burgk, J. L., Schmidt, T., Kaiser, V., Höning, K. & Hornung, V. A ligation-independent cloning technique for high-throughput assembly of transcription activator–like effector genes. Nat. Biotechnol. 31 , 76–81 (2013).

Ishino, Y., Shinagawa, H., Makino, K., Amemura, M. & Nakata, A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. J. Bacteriol. 169 , 5429–5433 (1987).

Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337 , 816–821 (2012).

Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology (Reading, England) 151 , 2551–2561 (2005).

Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading England) 151 , 653–663 (2005).

Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13 , 722–736 (2015).

Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9 , 467–477 (2011).

Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L. A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31 , 233–239 (2013).

Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507 , 62–67 (2014).

Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus . J. Bacteriol. 190 , 1390–1400 (2008).

Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109 , E2579–E2586 (2012).

Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8 , 2281–2308 (2013).

Doudna, J. A. & Charpentier, E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346 , 1258096 (2014).

Liu, C., Zhang, L., Liu, H. & Cheng, K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J. Control. Release. 266 , 17–26 (2017).

Biagioni, A. et al. Delivery systems of CRISPR/Cas9-based cancer gene therapy. J. Biol. Eng. 12 , 33 (2018).

Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature. 550 , 280–284 (2017).

Merkle, T. et al. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol . 37 , 133–138 (2019).

Tucker, B. A. et al. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice. PloS ONE 6 , e18992 (2011).

Homma, K. et al. Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors. Stem Cells (Dayton, OH) 31 , 1149–1159 (2013).

Cai, B. et al. Application of CRISPR/Cas9 technologies combined with iPSCs in the study and treatment of retinal degenerative diseases. Hum. Genet. 137 , 679–688 (2018).

Vogelstein, B. et al. Cancer genome landscapes. Science 339 , 1546–1558 (2013).

Choo, Y., Sánchez-García, I. & Klug, A. In vivo repression by a site-specific DNA-binding protein designed against an oncogenic sequence. Nature 372 , 642–645 (1994).

Do, T. U., Ho, B., Shih, S. J. & Vaughan, A. Zinc finger nuclease induced DNA double stranded breaks and rearrangements in MLL. Mutat. Res. 740 , 34–42 (2012).

Provasi, E. et al. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat. Med. 18 , 807–815 (2012).

Tanaka, A. et al. A novel therapeutic molecule against HTLV-1 infection targeting provirus. Leukemia 27 , 1621–1627 (2013).

Huang, N. et al. Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases. J. Exp. Clin. Cancer Res. 37 , 62 (2018).

Piganeau, M. et al. Cancer translocations in human cells induced by zinc finger and TALE nucleases. Genome Res. 23 , 1182–1193 (2013).

Puria, R., Sahi, S. & Nain, V. HER2+ breast cancer therapy: by CPP-ZFN mediated targeting of mTOR? Technol. Cancer Res. Treat. 11 , 175–180 (2012).

Herrmann, F. et al. p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid and validated by Solexa sequencing. PLoS ONE 6 , e20913 (2011).

Reik, A., Zhou, Yuanyue & Mendel, MatthewC. Zinc finger nucleases targeting the glucocorticoid receptor Allow IL-13 zetakine transgenic CTLs to kill glioblastoma cells in vivo in the presence of immunosuppressing glucocorticoids. Mol. Ther. 16 , S13–S14 (2008). D. E. P., 1 DiGiusto,2 & Jensen.

Google Scholar  

Marchiq, I., Le Floch, R., Roux, D., Simon, M. P. & Pouyssegur, J. Genetic disruption of lactate/H+ symporters (MCTs) and their subunit CD147/BASIGIN sensitizes glycolytic tumor cells to phenformin. Cancer Res. 75 , 171–180 (2015).

Miller, J. C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29 , 143–148 (2011).

Poirot, L. et al. Multiplex genome edited T-cell manufacturing platform for “off-the-shelf” adoptive T-cell immunotherapies. Cancer Res . 75 , 3853–3864 (2015).

Wang, J. et al. TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality. Sci. Rep. 5 , 18454 (2015).

Nyquist, M. D. et al. TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer. Proc. Natl Acad. Sci. USA 110 , 17492–17497 (2013).

Cai, Y. et al. Loss of chromosome 8p governs tumor progression and drug response by altering lipid metabolism. Cancer Cell 29 , 751–766 (2016).

Xiao, L. et al. LRH-1 drives hepatocellular carcinoma partially through induction of c-myc and cyclin E1, and suppression of p21. Cancer Manag. Res. 10 , 2389–2400 (2018).

Zhan, T., Rindtorff, N., Betge, J., Ebert, M. P. & Boutros, M. CRISPR/Cas9 for cancer research and therapy. Semin Cancer Biol. 55 , 106–119 (2019).

Heckl, D. et al. Generation of mouse models of myeloid malignancy with combinatorial genetic lesions using CRISPR-Cas9 genome editing. Nat. Biotechnol . 32 , 941–946 (2014).

Sánchez-Rivera, F. J. & Jacks, T. Applications of the CRISPR-Cas9 system in cancer biology. Nat. Rev. Cancer 15 , 387–395 (2015).

Sayin, V. I. & Papagiannakopoulos, T. Application of CRISPR-mediated genome engineering in cancer research. Cancer Lett . 387 , 10–17 (2016).

Matano, M. et al. Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat. Med. 21 , 256–262 (2015).

Roper, J. et al. Colonoscopy-based colorectal cancer modeling in mice with CRISPR-Cas9 genome editing and organoid transplantation. Nat. Protoc. 13 , 217–234 (2018).

Li, A. H. et al. Analysis of loss-of-function variants and 20 risk factor phenotypes in 8,554 individuals identifies loci influencing chronic disease. Nat. Genet. 47 , 640–642 (2015).

Abrahimi, P. et al. Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9. Circ. Res . 117 , 121–128 (2015).

Carroll, K. J. et al. A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9. Proc. Natl Acad. Sci. USA 113 , 338–343 (2016).

Willer, C. J. et al. Newly identified loci that influence lipid concentrations and risk of coronary artery disease. Nat. Genet. 40 , 161–169 (2008).

Gifford, C. A. et al. Oligogenic inheritance of a human heart disease involving a genetic modifier. Science 364 , 865–870 (2019).

Yang, D. et al. Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Res. 21 , 979–982 (2011).

Umeyama, K. et al. Generation of heterozygous fibrillin-1 mutant cloned pigs from genome-edited foetal fibroblasts. Sci. Rep. 6 , 24413 (2016).

Ang, Y. S. et al. Disease model of GATA4 mutation reveals transcription factor cooperativity in human cardiogenesis. Cell 167 , 1734–1749.e1722 (2016).

Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with induced pluripotent stem cell and heart-on-chip technologies. Nat. Med. 20 , 616–623 (2014).

Wallace, E. et al. Long QT syndrome: genetics and future perspective. Pediatr Cardiol. 40 , 1419–1430 (2019).

Burnett, J. R. & Hooper, A. J. PCSK9—a journey to cardiovascular outcomes. N. Engl. J. Med. 379 , 2161–2162 (2018).

Pollin, T. I. et al. A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 322 , 1702–1705 (2008).

Ding, Q. et al. Permanent alteration of PCSK9 with in vivo CRISPR-Cas9 genome editing. Circ. Res. 115 , 488–492 (2014).

Crosby, J. et al. Loss-of-function mutations in APOC3, triglycerides, and coronary disease. N. Engl. J. Med. 371 , 22–31 (2014).

Zhan, Y. et al. Establishment of a PRKAG2 cardiac syndrome disease model and mechanism study using human induced pluripotent stem cells. J. Mol. Cell. Cardiol . 117 , 49–61 (2018).

O’Rahilly, S. Human genetics illuminates the paths to metabolic disease. Nature 462 , 307–314 (2009).

Coppari, R. & Bjørbæk, C. Leptin revisited: its mechanism of action and potential for treating diabetes. Nat. Rev. Drug Disco. 11 , 692–708 (2012).

Giesbertz, P. et al. Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes. Diabetologia 58 , 2133–2143 (2015).

Chen, Y. et al. Generation of obese rat model by transcription activator-like effector nucleases targeting the leptin receptor gene. Sci. China Life Sci . 60 , 152–157(2016).

Bao, D. et al. Preliminary characterization of a leptin receptor knockout rat created by CRISPR/Cas9 system. Sci. Rep. 5 , 15942 (2015).

Wang, X. et al. Characterization of novel cytochrome P450 2E1 knockout rat model generated by CRISPR/Cas9. Biochem. Pharmacol. 105 , 80–90 (2016).

Claussnitzer, M. et al. FTO obesity variant circuitry and adipocyte browning in humans. N. Engl. J. Med . https://doi.org/10.1056/NEJMoa1502214 (2015).

Naylor, J. et al. Use of CRISPR/Cas-9 engineered INS-1 pancreatic beta cells to define the pharmacology of dual GIPR/GLP-1R agonists. Biochem. J . 473 , 2881–2891 (2016).

Vethe, H. et al. Probing the missing mature β-cell proteomic landscape in differentiating patient iPSC-derived cells. Sci. Rep. 7 , 4780 (2017).

Liao, H. K. et al. In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation. Cell 171 , 1495–1507.e15 (2017).

Tirronen, A., Hokkanen, K., Vuorio, T. & Ylä-Herttuala, S. Recent advances in novel therapies for lipid disorders. Hum. Mol. Genet . 28 , R49–R54 (2019).

Nakagawa, Y. et al. Hyperlipidemia and hepatitis in liver-specific CREB3L3 knockout mice generated using a one-step CRISPR/Cas9 system. Sci. Rep. 6 , 27857 (2016).

Carlson, D. F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl Acad. Sci. USA 109 , 17382–17387 (2012).

Burkhardt, R. et al. Trib1 is a lipid- and myocardial infarction-associated gene that regulates hepatic lipogenesis and VLDL production in mice. J. Clin. Invest. 120 , 4410–4414 (2010).

Nagiec, M. M. et al. Modulators of hepatic lipoprotein metabolism identified in a search for small-molecule inducers of tribbles pseudokinase 1 expression. PLoS ONE 10 , e0120295 (2015).

Nance, M. A. Genetics of Huntington disease. Handb. Clin. Neurol. 144 , 3–14 (2017).

Wood, L. B., Winslow, A. R. & Strasser, S. D. Systems biology of neurodegenerative diseases. Integr. Biol. (Camb.) 7 , 758–775 (2015).

Soto, C. & Pritzkow, S. Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat. Neurosci . 21 , 1332–1340 (2018).

Hu, Z., Yang, B., Mo, X. & Xiao, H. Mechanism and regulation of autophagy and its role in neuronal diseases. Mol. Neurobiol. 52 , 1190–1209 (2015).

Sahebkar, A., Panahi, Y., Yaribeygi, H. & Javadi, B. Oxidative stress in neurodegenerative diseases: a review. Mol Neurobiol. 53 , 4094–4125 (2018).

Rossi, F. & Cattaneo, E. Opinion: neural stem cell therapy for neurological diseases: dreams and reality. Nat. Rev. Neurosci. 3 , 401–409 (2002).

Fan, H. C. et al. The role of gene editing in neurodegenerative diseases. Cell Transpl. 27 , 364–378 (2018).

Article   Google Scholar  

Garriga-Canut, M. et al. Synthetic zinc finger repressors reduce mutant huntingtin expression in the brain of R6/2 mice. Proc. Natl Acad. Sci. USA 109 , E3136–E3145 (2012).

An, M. C. et al. Genetic correction of Huntington’s disease phenotypes in induced pluripotent stem cells. Cell Stem Cell 11 , 253–263 (2012).

Xu, X. et al. Reversal of phenotypic abnormalities by CRISPR/Cas9-mediated gene correction in Huntington disease patient-derived induced pluripotent stem cells. Stem Cell Rep. 8 , 619–633 (2017).

Yan, S. et al. A Huntingtin knockin pig model recapitulates features of selective neurodegeneration in Huntington’s disease. Cell 173 , 989–1002.e13 (2018).

Di Fede, G. et al. A recessive mutation in the APP gene with dominant-negative effect on amyloidogenesis. Science 323 , 1473–1477 (2009).

Jonsson, T. et al. A mutation in APP protects against Alzheimer’s disease and age-related cognitive decline. Nature 488 , 96–99 (2012).

Giaccone, G. et al. Neuropathology of the recessive A673V APP mutation: Alzheimer disease with distinctive features. Acta Neuropathol. 120 , 803–812 (2010).

Martiskainen, H. et al. Decreased plasma β-amyloid in the Alzheimer’s disease APP A673T variant carriers. Ann. Neurol . 82 ,128–132 (2017).

Wang, Y. et al. Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology. Sci. Rep. 6 , 22244 (2016).

Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533 , 125–129 (2016).

Abudayyeh, O. O. et al. A cytosine deaminase for programmable single-base RNA editing. Science 365 , 382–386 (2019).

Lubbe, S. & Morris, H. R. Recent advances in Parkinson’s disease genetics. J. Neurol. 261 , 259–266 (2014).

Dansithong, W., Paul, S., Scoles, D. R., Pulst, S. M. & Huynh, D. P. Generation of SNCA cell models using zinc finger nuclease (ZFN) technology for efficient high-throughput drug screening. PLoS ONE 10 , e0136930 (2015).

Reinhardt, P. et al. Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration to ERK-dependent changes in gene expression. Cell Stem Cell 12 , 354–367 (2013).

Soldner, F. et al. Parkinson-associated risk variant in distal enhancer of α-synuclein modulates target gene expression. Nature 533 , 95–99 (2016).

Chen, S., Yu, X. & Guo, D. CRISPR-Cas targeting of host genes as an antiviral strategy. Viruses 10 , 40 (2018).

Perez, E. E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26 , 808–816 (2008).

Hütter, G. et al. Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation. N. Engl. J. Med. 360 , 692–698 (2009).

Allers, K. et al. Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood 117 , 2791–2799 (2011).

Allen, A. G. et al. Gene editing of HIV-1 Co-receptors to prevent and/or cure virus infection. Front Microbiol 9 , 2940 (2018).

Didigu, C. A. et al. Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection. Blood 123 , 61–69 (2014).

Mussolino, C. et al. TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res. 42 , 6762–6773 (2014).

Ebina, H., Misawa, N., Kanemura, Y. & Koyanagi, Y. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus. Sci. Rep. 3 , 2510 (2013).

Mandal, P. K. et al. Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9. Cell Stem Cell 15 , 643–652 (2014).

Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol . 33 , 985–989 (2015).

Hu, W. et al. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection. Proc. Natl Acad. Sci. USA 111 , 11461–11466 (2014).

Moody, C. A. & Laimins, L. A. Human papillomavirus oncoproteins: pathways to transformation. Nat. Rev. Cancer 10 , 550–560 (2010).

Ding, W. et al. Zinc finger nucleases targeting the human papillomavirus E7 oncogene induce E7 disruption and a transformed phenotype in HPV16/18-positive cervical cancer cells. Clin. Cancer Res. 20 , 6495–6503 (2014).

Ren, C. et al. Zinc finger nuclease combines with cisplatin and trichostatin a enhances the antitumor potency in cervical cancer cells. Anticancer Agents Med. Chem. https://doi.org/10.2174/1871520618666180509152222 (2018).

Wayengera, M. Zinc finger arrays binding human papillomavirus types 16 and 18 genomic DNA: precursors of gene-therapeutics for in-situ reversal of associated cervical neoplasia. Theor. Biol. Med. Model 9 , 30 (2012).

Hu, Z. et al. TALEN-mediated targeting of HPV oncogenes ameliorates HPV-related cervical malignancy. J. Clin. Invest 125 , 425–436 (2015).

Zhen, S. et al. In vitro and in vivo synergistic therapeutic effect of cisplatin with human papillomavirus16 E6/E7 CRISPR/Cas9 on cervical cancer cell line. Transl. Oncol. 9 , 498–504 (2016).

Cradick, T. J., Keck, K., Bradshaw, S., Jamieson, A. C. & McCaffrey, A. P. Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol. Ther. 18 , 947–954 (2010).

Ramanan, V. et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci. Rep. 5 , 10833 (2015).

Wang, J. & Quake, S. R. RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection. Proc. Natl Acad. Sci. USA 111 , 13157–13162 (2014).

Cai, S. W., Zhang, Y., Hou, M. Z., Liu, Y. & Li, X. R. The research advances and applications of genome editing in hereditary eye diseases. Zhonghua Yan Ke Za Zhi 53 , 386–391 (2017).

Bjork, S., Hurt, C. M., Ho, V. K. & Angelotti, T. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity. PloS ONE 8 , e76366 (2013).

Arno, G. et al. Mutations in REEP6 cause autosomal-recessive retinitis pigmentosa. Am. J. Hum. Genet. 99 , 1305–1315 (2016).

Bowes, C. et al. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature 347 , 677–680 (1990).

Keeler, C. E. THE geotropic reaction of rodless mice in light and in darkness. J. Gen. Physiol. 11 , 361–368 (1928).

Wu, W. H. et al. CRISPR repair reveals causative mutation in a preclinical model of retinitis pigmentosa. Mol. Ther.: J. Am. Soc. Gene Ther. 24 , 1388–1394 (2016).

McGill, T. J. et al. Optomotor and immunohistochemical changes in the juvenile S334ter rat. Exp. Eye Res. 104 , 65–73 (2012).

Cho, G. Y. et al. CRISPR-mediated ophthalmic genome surgery. Curr. Ophthalmol. Rep. 5 , 199–206 (2017).

Bakondi, B. et al. In Vivo CRISPR/Cas9 gene editing corrects retinal dystrophy in the S334ter-3 rat model of autosomal dominant retinitis pigmentosa. Mol. Ther.: J. Am. Soc. Gene Ther. 24 , 556–563 (2016).

Latella, M. C. et al. In vivo editing of the human mutant rhodopsin gene by electroporation of plasmid-based CRISPR/Cas9 in the mouse retina. Mol. Ther. Nucleic Acids 5 , e389 (2016).

Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540 , 144–149 (2016).

Bassuk, A. G., Zheng, A., Li, Y., Tsang, S. H. & Mahajan, V. B. Precision medicine: genetic repair of retinitis pigmentosa in patient-derived stem cells. Sci. Rep. 6 , 19969 (2016).

Deng, W. L. et al. Gene correction reverses ciliopathy and photoreceptor loss in iPSC-derived retinal organoids from retinitis pigmentosa patients. Stem Cell Rep. 10 , 1267–1281 (2018).

Liao, C., Zhang, D., Mungo, C., Tompkins, D. A. & Zeidan, A. M. Is diabetes mellitus associated with increased incidence and disease-specific mortality in endometrial cancer? A systematic review and meta-analysis of cohort studies. Gynecologic Oncol. 135 , 163–171 (2014).

Zhong, H., Chen, Y., Li, Y., Chen, R. & Mardon, G. CRISPR-engineered mosaicism rapidly reveals that loss of Kcnj13 function in mice mimics human disease phenotypes. Sci. Rep. 5 , 8366 (2015).

Ruan, G. X. et al. CRISPR/Cas9-mediated genome editing as a therapeutic approach for leber congenital amaurosis 10. Mol. Ther.: J. Am. Soc. Gene Ther. 25 , 331–341 (2017).

Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25 , 229–233 (2019).

Lee, W. H., Murphree, A. L. & Benedict, W. F. Expression and amplification of the N-myc gene in primary retinoblastoma. Nature 309 , 458–460 (1984).

Solin, S. L., Shive, H. R., Woolard, K. D., Essner, J. J. & McGrail, M. Rapid tumor induction in zebrafish by TALEN-mediated somatic inactivation of the retinoblastoma1 tumor suppressor rb1. Sci. Rep. 5 , 13745 (2015).

Naert, T. et al. CRISPR/Cas9 mediated knockout of rb1 and rbl1 leads to rapid and penetrant retinoblastoma development in Xenopus tropicalis. Sci. Rep. 6 , 35264 (2016).

Tu, J. et al. Generation of human embryonic stem cell line with heterozygous RB1 deletion by CRIPSR/Cas9 nickase. Stem Cell Res 28 , 29–32 (2018).

Hollands, H. et al. Do findings on routine examination identify patients at risk for primary open-angle glaucoma? The rational clinical examination systematic review. Jama 309 , 2035–2042 (2013).

Alward, W. L. et al. Clinical features associated with mutations in the chromosome 1 open-angle glaucoma gene (GLC1A). New Engl. J. Med. 338 , 1022–1027 (1998).

Stone, E. M. et al. Identification of a gene that causes primary open angle glaucoma. Science 275 , 668–670 (1997).

Kim, B. S. et al. Targeted disruption of the myocilin gene (Myoc) suggests that human glaucoma-causing mutations are gain of function. Mol. Cell. Biol. 21 , 7707–7713 (2001).

Carbone, M. A. et al. Overexpression of myocilin in the Drosophila eye activates the unfolded protein response: implications for glaucoma. PloS ONE 4 , e4216 (2009).

Joe, M. K. et al. Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells. Biochem. Biophys. Res. Commun. 312 , 592–600 (2003).

Liu, Y. & Vollrath, D. Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma. Hum. Mol. Genet. 13 , 1193–1204 (2004).

Yam, G. H., Gaplovska-Kysela, K., Zuber, C. & Roth, J. Aggregated myocilin induces russell bodies and causes apoptosis: implications for the pathogenesis of myocilin-caused primary open-angle glaucoma. Am. J. Pathol. 170 , 100–109 (2007).

Jain, A. et al. CRISPR-Cas9-based treatment of myocilin-associated glaucoma. Proc. Natl Acad. Sci. USA 114 , 11199–11204 (2017).

Liao, H. et al. Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy. PloS ONE 6 , e28582 (2011).

Courtney, D. G. et al. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Ther. 23 , 108–112 (2016).

Shima, M. et al. Factor VIII-mimetic function of humanized bispecific antibody in hemophilia A. N. Engl. J. Med. 374 , 2044–2053 (2016).

Park, C. Y. et al. Functional correction of large factor VIII gene chromosomal inversions in hemophilia a patient-derived iPSCs using CRISPR-Cas9. Cell Stem Cell 17 , 213–220 (2015).

Guan, Y. et al. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med. 8 , 477–488 (2016).

Hai, T., Teng, F., Guo, R., Li, W. & Zhou, Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Res. 24 , 372–375 (2014).

Rees, D. C., Williams, T. N. & Gladwin, M. T. Sickle-cell disease. Lancet 376 , 2018–2031 (2010).

Dever, D. P. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539 , 384–389 (2016).

Martin, R. M. et al. Highly efficient and marker-free genome editing of human pluripotent stem cells by CRISPR-Cas9 RNP and AAV6 donor-mediated homologous recombination. Cell Stem Cell 24 , 821–828.e825 (2019).

Verhaart, I. E. C. & Aartsma-Rus, A. Therapeutic developments for Duchenne muscular dystrophy. Nat. Rev. Neurol. 15 , 373–386 (2019).

Larcher, T. et al. Characterization of dystrophin deficient rats: a new model for Duchenne muscular dystrophy. PLoS ONE 9 , e110371 (2014).

Sui, T. et al. A novel rabbit model of Duchenne muscular dystrophy generated by CRISPR/Cas9. Dis. Model Mech . 11 , dmm.032201 (2018).

Chen, Y. et al. Generation of cynomolgus monkey chimeric fetuses using embryonic stem cells. Cell Stem Cell 17 , 116–124 (2015).

Chen, Y. et al. Functional disruption of the dystrophin gene in rhesus monkey using CRISPR/Cas9. Hum. Mol. Genet. 24 , 3764–3774 (2015).

Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol. Ther . 24 , 564–569 (2015).

Bengtsson, N. E. et al. Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nat. Commun. 8 , 14454 (2017).

Nelson, C. E. et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 351 , 403–407 (2016).

Pai, S. Y. et al. Transplantation outcomes for severe combined immunodeficiency, 2000-2009. N. Engl. J. Med. 371 , 434–446 (2014).

Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25 , 1298–1306 (2007).

Flisikowska, T. et al. Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases. PLoS ONE 6 , e21045 (2011).

Wang, Y. et al. Generation of knockout rabbits using transcription activator-like effector nucleases. Cell Regen. (Lond.) 3 , 3 (2014).

Yan, Q. et al. Generation of multi-gene knockout rabbits using the Cas9/gRNA system. Cell Regen. (Lond.) 3 , 12 (2014).

Zhou, J. et al. One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Int. J. Biochem. Cell Biol. 46 , 49–55 (2014).

Ott de Bruin, L. M., Volpi, S. & Musunuru, K. Novel genome-editing tools to model and correct primary immunodeficiencies. Front Immunol. 6 , 250 (2015).

Chen, Y. et al. Modeling Rett syndrome using TALEN-Edited MECP2 mutant cynomolgus monkeys. Cell 169 , 945–955.e910 (2017).

Chen, J. R. et al. Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation. Cell Death Differ . 9 , eaaj2013 (2016).

Jiang, W. et al. Production of Wilson disease model rabbits with homology-directed precision point mutations in the ATP7B gene using the CRISPR/Cas9 system. Sci. Rep. 8 , 1332 (2018).

Kurome, M. et al. 361 growth hormone receptor mutant pigs produced by using the clustered regularly interspaced short palindromic repeats (crispr) and crispr-associated systems in in vitro-produced zygotes. Reprod. Fertil. Dev. 27 , 269 (2014).

Tseng, W. C. et al. Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds. Dis. Model Mech. 11 , dmm.034165 (2018).

Abdul-Wahab, A., Qasim, W. & McGrath, J. A. Gene therapies for inherited skin disorders. Semin Cutan. Med. Surg. 33 , 83–90 (2014).

Kirkwood, J. M. et al. Immunotherapy of cancer in 2012. CA Cancer J. Clin. 62 , 309–335 (2012).

Rein, L. A. M., Yang, H. & Chao, N. J. Applications of gene editing technologies to cellular therapies. Biol. Blood Marrow Transpl. 24 , 1537–1545 (2018).

June, C. H. & Sadelain, M. Chimeric antigen receptor therapy. N. Engl. J. Med 379 , 64–73 (2018).

Sadelain, M., Brentjens, R. & Rivière, I. The promise and potential pitfalls of chimeric antigen receptors. Curr. Opin. Immunol. 21 , 215–223 (2009).

Davenport, A. J. et al. CAR-T cells inflict sequential killing of multiple tumor target cells. Cancer Immunol. Res 3 , 483–494 (2015).

Zhao, J., Lin, Q., Song, Y. & Liu, D. Universal CARs, universal T cells, and universal CAR T cells. J. Hematol. Oncol. 11 , 132 (2018).

Torikai, H. et al. A foundation for universal T-cell based immunotherapy: T cells engineered to express a CD19-specific chimeric-antigen-receptor and eliminate expression of endogenous TCR. Blood 119 , 5697–5705 (2012).

Torikai, H. et al. Toward eliminating HLA class I expression to generate universal cells from allogeneic donors. Blood 122 , 1341–1349 (2013).

Qasim, W. et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci. Transl. Med . 9 , eaaj2013 (2017).

Wei, G., Wang, J., Huang, H. & Zhao, Y. Novel immunotherapies for adult patients with B-lineage acute lymphoblastic leukemia. J. Hematol. Oncol. 10 , 150 (2017).

Liu, X. et al. CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells. Cell Res . 27 , 154–157 (2017).

Ren, J. et al. A versatile system for rapid multiplex genome-edited CAR T cell generation. Oncotarget 8 , 17002–17011 (2017).

Kenderian, S. S. et al. Identification of PD1 and TIM3 as checkpoints that limit chimeric antigen receptor T cell efficacy in leukemia. Biol. Blood Marrow Transplant. 22 , S19–S21 (2016).

Leone, R. D. & Emens, L. A. Targeting adenosine for cancer immunotherapy. J. Immunother. Cancer 6 , 57 (2018).

Zhang, Y. et al. CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells. Front Med . 11 , 554–562 (2017).

Wong, A. S. et al. Multiplexed barcoded CRISPR-Cas9 screening enabled by CombiGEM. Proc. Natl Acad. Sci. USA . 113 , 2544–2549 (2016).

Yu, J. S. L. & Yusa, K. Genome-wide CRISPR-Cas9 screening in mammalian cells. Methods. 164–165 , 29–35 (2019).

Luo, J. CRISPR/Cas9: from genome engineering to cancer drug discovery. Trends . Cancer 2 , 313–324 (2016).

Bester, A. C. et al. An integrated genome-wide CRISPRa approach to functionalize lncRNAs in drug resistance. Cell 173 , 649–664.e620 (2018).

Tzelepis, K. et al. A CRISPR dropout screen identifies genetic vulnerabilities and therapeutic targets in acute myeloid leukemia. Cell Rep. 17 , 1193–1205 (2016).

Munoz, D. M. et al. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer Discov 6 . 900–13. (2016).

Hart, T. et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell 163 , 1515–1526 (2015).

Wang, T. et al. Gene essentiality profiling reveals gene networks and synthetic lethal interactions with oncogenic Ras. Cell 168 , 890–903.e15 (2017).

Chen, S. et al. Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis. Cell 160 , 1246–1260 (2015).

McFadden, D. G. et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 156 , 1298–1311 (2014).

Tang, J. T. et al. MicroRNA 345, a methylation-sensitive microRNA is involved in cell proliferation and invasion in human colorectal cancer. Carcinogenesis 32 , 1207–1215 (2011).

Cheng, Z., Ma, R., Tan, W. & Zhang, L. MiR-152 suppresses the proliferation and invasion of NSCLC cells by inhibiting FGF2. Exp. Mol. Med. 46 , e112 (2014).

Song, C. Q. et al. Genome-wide CRISPR Screen Identifies Regulators of MAPK as Suppressors of Liver Tumors in Mice. Gastroenterology 5 , 1161–1173.e1 (2016).

Han, K. et al. Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions. Nat. Biotechnol 35 , 463–474. (2017).

Liu, S. J. et al. CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells. Science 355 , eaah7111 (2016).

Zhu, S. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library. Nat. Biotechnol . 34 , 1279–1286 (2016).

Esposito, R. et al. Hacking the cancer genome: profiling therapeutically actionable long non-coding RNAs using CRISPR-Cas9 screening. Cancer Cell 35 , 545–557 (2019).

Boettcher, M. et al. Dual gene activation and knockout screen reveals directional dependencies in genetic networks. Nat. Biotechnol. 36 , 170–178 (2018).

Yuan, Z. et al. Olaparib hydroxamic acid derivatives as dual PARP and HDAC inhibitors for cancer therapy. Bioorg. Med. Chem . 25 , 4100–4109 (2017).

Shen, J. P. et al. Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions. Nat. Methods 14 , 573–576 (2017).

Baliou, S. et al. CRISPR therapeutic tools for complex genetic disorders and cancer (Review). Int. J. Oncol 53 . 443–468 (2018).

Shalem, O., Sanjana, N. E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16 , 299–311 (2015).

Heigwer, F. et al. CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries. Genome Biol. 17 , 55 (2016).

Ruiz, S. et al. A Genome-wide CRISPR Screen Identifies CDC25A as a determinant of sensitivity to ATR inhibitors. Mol. Cell 62 , 307–313 (2016).

Krall, E. B. et al. KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer. Elife 6 , e18970 (2017).

Manguso, R. T. et al. In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target. Nature 547 , 413–418 (2017).

Flynn, R. et al. CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells. Exp. Hematol. 43 , 838–848.e833 (2015).

Rahman, N. Mainstreaming genetic testing of cancer predisposition genes. Clin. Med 14 , 436–439 (2014).

Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356 , 438–442 (2017).

Chen, J. S. et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science https://doi.org/10.1126/science.aar6245 (2018).

Chertow, D. S. Next-generation diagnostics with CRISPR. Science 360 , 381–382 (2018).

Yang, H. et al. Break breast cancer addiction by CRISPR/Cas9 genome editing. J. Cancer 9 , 219–231 (2018).

Wang, Y. et al. The BRCA1-Δ11q alternative splice isoform bypasses germline mutations and promotes therapeutic resistance to PARP inhibition and cisplatin. Cancer Res. 76 , 2778–2790 (2016).

Cyranoski, D. CRISPR gene-editing tested in a person for the first time. Nature 539 , 479 (2016).

Lázaro, C., Ravella, A., Gaona, A., Volpini, V. & Estivill, X. Neurofibromatosis type 1 due to germ-line mosaicism in a clinically normal father. N. Engl. J. Med. 331 , 1403–1407 (1994).

Wilen, C. B. et al. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases. PLoS Pathog. 7 , e1002020 (2011).

Lunzen, J. V. et al. Transfer of autologous gene-modified T cells in HIV-infected patients with advanced immunodeficiency and drug-resistant virus. Mol. Ther. 15 , 1024–1033 (2007).

Voit, R. A., McMahon, M. A., Sawyer, S. L. & Porteus, M. H. Generation of an HIV resistant T-cell line by targeted “stacking” of restriction factors. Mol. Ther. 21 , 786–795 (2013).

Tebas, P. et al. Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV. N. Engl. J. Med. 370 , 901–910 (2014).

Xu, L. et al. CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N. Engl. J. Med . 381 ,1240–1247 (2019).

Choi, K. D. et al. Hematopoietic and endothelial differentiation of human induced pluripotent stem cells. Stem Cells 27 , 559–567 (2009).

Dolan, G. et al. Haemophilia B: Where are we now and what does the future hold? Blood Rev. 32 , 52–60 (2018).

Origa, R. Beta-thalassemia. Genet Med 19 , 609–619 (2017).

Bauer, D. E. & Orkin, S. H. Hemoglobin switching’s surprise: the versatile transcription factor BCL11A is a master repressor of fetal hemoglobin. Curr. Opin. Genet. Dev. 33 , 62–70 (2015).

Moore, C. B. T., Christie, K. A., Marshall, J. & Nesbit, M. A. Personalised genome editing—the future for corneal dystrophies. Prog. Retin Eye Res. 65 , 147–165 (2018).

Wen, W. S., Yuan, Z. M., Ma, S. J., Xu, J. & Yuan, D. T. CRISPR-Cas9 systems: versatile cancer modelling platforms and promising therapeutic strategies. Int. J. Cancer 138 , 1328–1336 (2016).

Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31 , 822–826 (2013).

Guilinger, J. P. et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat. Methods 11 , 429–435 (2014).

Pattanayak, V., Ramirez, C. L., Joung, J. K. & Liu, D. R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8 , 765–770 (2011).

Vouillot, L., Thélie, A. & Pollet, N. Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 5 , 407–415 (2015).

Article   CAS   PubMed Central   Google Scholar  

Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31 , 839–843 (2013).

Seeger, C. & Sohn, J. A. Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA. Mol. Ther 24 , 1258–1266 (2016).

Gabriel, R. et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29 , 816–823 (2011).

Osborn, M. J. et al. Evaluation of TCR Gene Editing achieved by TALENs, CRISPR/Cas9 and megaTAL nucleases. Mol. Ther. 24 , 570–581(2015).

Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31 , 827–832 (2013).

Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31 , 833–838 (2013).

Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33 , 139–142 (2015).

Moon, S. B., Kim, D. Y., Ko, J. H., Kim, J. S. & Kim, Y. S. Improving CRISPR Genome Editing by Engineering Guide RNAs. Trends Biotechnol 37 , 870–882 (2019).

Cho, S. W. et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24 , 132–141 (2014).

Xie, S., Shen, B., Zhang, C., Huang, X. & Zhang, Y. sgRNAcas9: a software package for designing CRISPR sgRNA and evaluating potential off-target cleavage sites. PLoS ONE 9 , e100448 (2014).

Sander, J. D. et al. ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool. Nucleic Acids Res. 38 , W462–W468 (2010).

Kiani, S. et al. CRISPR transcriptional repression devices and layered circuits in mammalian cells. Nat. Methods 11 , 723–726 (2014).

Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351 , 84-88 (2015).

Kleinstiver, B. P. et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature https://doi.org/10.1038/nature16526 (2016).

Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24 , 1012–1019 (2014).

Suresh, B., Ramakrishna, S. & Kim, H. Cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA for genome editing. Methods Mol. Biol. 1507 , 81–94 (2017).

Dong, L. et al. An anti-CRISPR protein disables type V Cas12a by acetylation. Nat. Struct. Mol. Biol. 26 , 308–314 (2019).

Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci. Adv. 3 , e1701620 (2017).

Shrivastav, M., De Haro, L. P. & Nickoloff, J. A. Regulation of DNA double-strand break repair pathway choice. Cell Res. 18 , 134–147 (2008).

Ciccia, A. & Elledge, S. J. The DNA damage response: making it safe to play with knives. Mol. Cell 40 , 179–204 (2010).

Chapman, J. R., Taylor, M. R. & Boulton, S. J. Playing the end game: DNA double-strand break repair pathway choice. Mol. Cell 47 , 497–510 (2012).

Maruyama, T. et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat. Biotechnol. 33 , 538–542 (2015).

Chu, V. T. et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat. Biotechnol . 33 , 543–548 (2015).

Canny, M. D. et al. Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat. Biotechnol . 36 , 95–102 (2017).

Song, J. et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat. Commun. 7 , 10548 (2016).

Gao, Y. et al. Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 404 , 897–900 (2000).

Moshous, D. et al. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell 105 , 177–186 (2001).

Shah, S. Z. et al. Advances In research on genome editing Crispr-Cas9 technology. J. Ayub Med Coll. Abbottabad 31 , 108–122 (2019).

PubMed   Google Scholar  

Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. Elife 3 , e04766 (2014).

Kim, K. et al. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35 , 435–437 (2017).

Zafra, M. P. et al. Optimized base editors enable efficient editing in cells, organoids and mice. Nat. Biotechnol. 36 , 888–893 (2018).

Grünewald, J. et al. CRISPR DNA base editors with reduced RNA off-target and self-editing activities. Nat. Biotechnol. 37 , 1041–1048 (2019).

Article   PubMed   CAS   PubMed Central   Google Scholar  

Zong, Y. et al. Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat. Biotechnol. 36 , 950–953 (2018).

Hur, J. K. et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat. Biotechnol. 34 , 807–808 (2016).

Gori, J. L. et al. Delivery and Specificity of CRISPR-Cas9 Genome Editing Technologies for Human Gene Therapy. Hum. Gene Ther . https://doi.org/10.1089/hum.2015.074 (2015).

Zuris, J. A. et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33 , 73–80 (2015).

Mout, R., Ray, M., Lee, Y. W., Scaletti, F. & Rotello, V. M. In vivo delivery of CRISPR/Cas9 for therapeutic gene editing: progress and challenges. Bioconjug. Chem . 28 , 880–884 (2017).

Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Disco. 16 , 387–399 (2017).

Kotterman, M. A. & Schaffer, D. V. Engineering adeno-associated viruses for clinical gene therapy. Nat. Rev. Genet. 15 , 445–451 (2014).

Maggio, I. et al. Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells. Sci. Rep. 4 , 5105 (2014).

Feng, M. et al. Stable in vivo gene transduction via a novel adenoviral/retroviral chimeric vector. Nat. Biotechnol. 15 , 866–870 (1997).

Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32 , 267–273 (2014).

Paulk, N. K. et al. Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51 , 1200–1208 (2010).

Charlesworth, C. T. et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. bioRxiv https://doi.org/10.1101/243345 (2018).

Ihry, R. J. et al. p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat. Med . 24 , 939–946 (2018).

Liang, C. et al. Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma. Biomaterials 147 , 68–85 (2017).

Luo, Y. L. et al. Macrophage-specific in vivo gene editing using cationic lipid-assisted polymeric nanoparticles. ACS Nano 12 , 994–1005 (2018).

Finn, J. D. et al. A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22 , 2227–2235 (2018).

Wang, H. X. et al. Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide. Proc. Natl Acad. Sci. USA 115 , 4903–4908 (2018).

Ramakrishna, S. et al. Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA. Genome Res. 24 , 1020–1027 (2014).

Ma, Y. et al. Increasing the efficiency of CRISPR/Cas9-mediated precise genome editing in rats by inhibiting NHEJ and using Cas9 protein. RNA Biol. 13 , 605–612 (2016).

Guenther, C. M. et al. Synthetic virology: engineering viruses for gene delivery. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 6 , 548–558 (2014).

Cronin, J., Zhang, X. Y. & Reiser, J. Altering the tropism of lentiviral vectors through pseudotyping. Curr. Gene Ther. 5 , 387–398 (2005).

Ho, M. L. et al. Efficiency of protease-activatable virus nanonodes tuned through incorporation of wild-type capsid subunits. Cell. Mol. Bioeng. 7 , 334–343 (2014).

Asuri, P. et al. Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells. Mol. Ther. 20 , 329–338 (2012).

Hofherr, S. E., Mok, H., Gushiken, F. C., Lopez, J. A. & Barry, M. A. Polyethylene glycol modification of adenovirus reduces platelet activation, endothelial cell activation, and thrombocytopenia. Hum. Gene Ther. 18 , 837–848 (2007).

Lee, G. K., Maheshri, N., Kaspar, B. & Schaffer, D. V. PEG conjugation moderately protects adeno-associated viral vectors against antibody neutralization. Biotechnol. Bioeng. 92 , 24–34 (2005).

Kay, M. A., He, C. Y. & Chen, Z. Y. A robust system for production of minicircle DNA vectors. Nat. Biotechnol. 28 , 1287–1289 (2010).

Koo, T. et al. Selective disruption of an oncogenic mutant allele by CRISPR/Cas9 induces efficient tumor regression. Nucleic Acids Res . 45 , 7897–7908 (2017).

Lee, K. et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 1 , 889–901 (2017).

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Acknowledgements

This work is supported by the National Natural Science Foundation of China (No. 81602492), the National Key Research and Development Program of China (No. 2016YFA0201402) and the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (No. 2018ZX09733001).

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These authors contributed equally: Hongyi Li, Yang Yang

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Department of Gynecology and Obstetrics, Development and Related Disease of Women and Children Key Laboratory of Sichuan Province, Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, West China Second Hospital, Sichuan University, Chengdu, 610041, P. R. China

Hongyi Li, Yang Yang & Xia Zhao

Laboratory of Aging Research and Cancer Drug Target, State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu, Sichuan, 610041, P. R. China

Weiqi Hong & Mengyuan Huang

Department of Biomedical Sciences, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND, 58203, USA

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Correspondence to Min Wu or Xia Zhao .

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Li, H., Yang, Y., Hong, W. et al. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Sig Transduct Target Ther 5 , 1 (2020). https://doi.org/10.1038/s41392-019-0089-y

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CRISPR-mediated genome editing and human diseases

Affiliations.

• 1 Marshall Institute for Interdisciplinary Research (MIIR), Marshall University, Weisberg Engineering Complex, Marshall University, 1628 Third Avenue, Huntington, WV 25703, USA.
• 2 University of Texas Health Science Center at San Antonio, Department of Medicine, Division of Geriatrics, Gerontology, and Palliative Medicine, San Antonio, TX 78229, USA.
• 3 GRECC, South Texas VA Healthcare System, San Antonio, TX 78229, USA.
• 4 Committee on Immunology, Section of Rheumatology, Department of Medicine, Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637, USA.
• PMID: 30258895
• PMCID: PMC6150104
• DOI: 10.1016/j.gendis.2016.07.003

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has emerged as a powerful technology for genome editing and is now widely used in basic biomedical research to explore gene function. More recently, this technology has been increasingly applied to the study or treatment of human diseases, including Barth syndrome effects on the heart, Duchenne muscular dystrophy, hemophilia, β-Thalassemia, and cystic fibrosis. CRISPR/Cas9 (CRISPR-associated protein 9) genome editing has been used to correct disease-causing DNA mutations ranging from a single base pair to large deletions in model systems ranging from cells in vitro to animals in vivo . In addition to genetic diseases, CRISPR/Cas9 gene editing has also been applied in immunology-focused applications such as the targeting of C-C chemokine receptor type 5, the programmed death 1 gene, or the creation of chimeric antigen receptors in T cells for purposes such as the treatment of the acquired immune deficiency syndrome (AIDS) or promoting anti-tumor immunotherapy. Furthermore, this technology has been applied to the genetic manipulation of domesticated animals with the goal of producing biologic medical materials, including molecules, cells or organs, on a large scale. Finally, CRISPR/Cas9 has been teamed with induced pluripotent stem (iPS) cells to perform multiple tissue engineering tasks including the creation of disease models or the preparation of donor-specific tissues for transplantation. This review will explore the ways in which the use of CRISPR/Cas9 is opening new doors to the treatment of human diseases.

Keywords: CRISPR; DNA double-stranded break; Genome editing; Human diseases; iPS cells.

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• Jansen R., Embden J.D., Gaastra W., Schouls L.M. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002;43(6):1565–1575. - PubMed
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Genome Editing

Gene scissors, molecular scalpel – these descriptive terms are intended to convey what the new method of gene editing with rather unwieldy name of CRISPR/Cas9 can do. As they suggest, the system, which, in its natural form, consists of two RNA molecules and one protein molecule, can cleave the hereditary molecule DNA. Moreover, it can do this with surgical precision at a specific site in the genome. This enables researchers to switch genes off or insert new sequences at the cutting site. As a result, DNA can be modified much faster and more easily than was possible using previous gene-editing methods. Although the system basically sounds simple, various factors must be coordinated with extreme precision for the gene scissors to be able to function with such accuracy. For this reason, even after 30 years of research, the functioning of CRISPR/Cas9 is still not entirely understood.

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Treatment of Genetic Diseases With CRISPR Genome Editing

• 1 Division of Allergy, Immunology, and Bone Marrow Transplantation, Department of Pediatrics, University of California, San Francisco
• 2 Pediatric Scientist Development Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, San Francisco, California
• 3 Innovative Genomics Institute, University of California, Berkeley
• 4 Department of Molecular and Cell Biology, Department of Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California, Berkeley
• 5 Howard Hughes Medical Institute, University of California, Berkeley
• 6 Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, California
• 7 Gladstone-UCSF Institute of Genomic Immunology, San Francisco, California
• Viewpoint Genomic Engineering and the Future of Medicine Jennifer A. Doudna, PhD JAMA
• Viewpoint Gene Editing Using CRISPR Anthony L. Komaroff, MD JAMA
• Medical News & Perspectives With First CRISPR Trials, Gene Editing Moves Toward the Clinic Tracy Hampton, PhD JAMA
• Bench to Bedside Virus Surveillance and Diagnosis With a CRISPR-Based Platform Tracy Hampton, PhD JAMA
• Medical News & Perspectives After the First Pig-to-Human Heart Transplant, Scientists Look to the Future of Cardiac Xenotransplantation Jyoti Madhusoodanan JAMA
• Medical News & Perspectives Highlights From the American Heart Association’s Scientific Sessions 2022—COVID-19’s Ripple Effects, a Triglyceride Disappointment, Gene Editing Advances, and More Jennifer Abbasi JAMA
• Biotech Innovations Researchers Use Plasmid Gene Editing to Personalize Solid Tumor T-Cell Therapy Howard Larkin JAMA
• Biotech Innovations “Off-the-Shelf” CAR T-Cell Therapy Tested in Pediatric B-Cell Leukemia Howard Larkin JAMA
• Viewpoint The CRISPR Patent Ruling and Implications for Medicine Jordan Paradise, JD JAMA
• Medical News in Brief Stem Cell Gene Editing Improved Sickle Cell Disease Outcomes Emily Harris JAMA

In nature, microorganisms use CRISPR (clustered regularly interspaced palindromic repeats) and CRISPR-associated (Cas) proteins for antiviral immunity through recognition and destruction of specific DNA sequences. Over the past decade, CRISPR genome editing has been developed to create transformative technologies to treat, cure, and prevent human disease.

CRISPR genome editing allows scientists to change DNA sequences in cells at virtually any desired position, enabling both fundamental research and therapeutic applications ( Figure ). CRISPR-Cas9, the most widely used genome editor, is an RNA-guided DNA-cutting enzyme that makes double-stranded DNA breaks at preselected (on-target) positions in the DNA of living cells. 1 Repair of the break site results in either small insertions and deletions ( indels ) introduced by error-prone repair or insertion of a new DNA donor sequence chosen by the investigator (homology-directed repair). Indels are useful for interrupting gene function, whereas sequence insertion can replace a defective sequence to restore gene function. 2 In either case, controlling the exact editing outcome for a particular indication in a specific cell type or organ is challenging, and unintended (off-target) DNA changes can be harmful.

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Kan MJ , Doudna JA. Treatment of Genetic Diseases With CRISPR Genome Editing. JAMA. 2022;328(10):980–981. doi:10.1001/jama.2022.13468

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Faculty of Health and Medical Sciences

Genome Editing Laboratory

Welcome to the Genome Editing Laboratory, where we are using state-of-the-art molecular genetic approaches to develop and improve CRISPR genome editing technology. Our mission is to enhance human health by harnessing the power of CRISPR to cure genetic diseases, understand the pathology of common genetic diseases, control invasive mouse populations, and continuously improve CRISPR gene editing technology.

CRISPR genome editing technology is revolutionizing medicine, biology, and agriculture. It enables targeted genetic modification of virtually any species with unprecedented efficiency. Since the first CRISPR editing publication in late 2012, it has had an enormous impact on basic and applied research. Our laboratory is focused on using CRISPR technology to understand the pathology of common genetic diseases by generating CRISPR mice that model disease-causing mutations in humans. We are also developing approaches to cure genetic diseases, such as DMD, retinitis pigmentosa and PCDH19-clustering epilepsy, by correcting disease-causing mutations in vivo. At the same time, we are continuously striving to improve the CRISPR gene editing technology to make it more precise and efficient. In addition, we are leading the world in the development of CRISPR gene drives in mice. This powerful technology has enormous potential for controlling invasive mouse populations that spread (zoonotic) disease, cause species extinction, and reduce agricultural productivity. At the Genome Editing Laboratory, we are committed to using the latest technological advances to improve human health and welfare. Our team of dedicated scientists is at the forefront of CRISPR research, striving to push the boundaries of what is possible in this exciting field. The Genome Editing Laboratory hosts the Australia Gene Editing Network (AGENt), a community that brings together Australian researchers interested in gene editing. AGENt provides networking opportunities and resources for researchers from all levels and fields to collaborate and accelerate gene editing research. AGENt also provides regular seminar series featuring speakers from across Australia. To join AGENt, individuals should fill in the following Google Form  https://forms.gle/6HqpA6jCpZYquoCQA  or contact Dr Fatwa Adikusuma at  [email protected] .

Lead researcher

Professor Paul Thomas

Professor Paul Thomas leads the Genome Editing Laboratory and is Director of the SA Genome Editing (SAGE) Facility at the University of Adelaide and SAHMRI. He has over 20 years experience in generation and analysis of mouse models for neurodevelopmental diseases including intellectual disability and epilepsy. Professor Thomas has published >80 research articles and has considerable expertise in CRISPR/Cas genome editing through generation of >60 genetically modified mouse lines including first generation CRISPR gene drives.

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Genome Editing and Biological Weapons

Assessing the Risk of Misuse

• © 2023
• Katherine Paris 0

George Mason University, Arlington, USA

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Introduces to the state-of-the-art genome editing techniques CRISPR, TALEN and ZFN

Assesses the risk of misuse of genome editing technologies in a comprehensive manner

Discusses applications of genome editing techniques in bioweapon development

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About this book

This monograph introduces current genome editing technologies—clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs)—and provides an assessment of the risk of misuse of these technologies based on the following parameters: accessibility, ease of misuse, magnitude of potential harm, and imminence of potential misuse. The findings from this assessment are applied to analyze and evaluate the threat posed by the intentional misuse of genome editing technologies to develop biological weapons.  Furthermore, the book discusses the implications of misuse for different applications of genome editing, such as making existing pathogens more dangerous, modifying the human microbiome, weaponizing gene drives, engineering super soldiers, and augmenting the general population to confer economic advantages.   

The book provides a comprehensive assessment of how feasible it is for users with different levels of knowledge and skill to acquire and then to apply the technologies to develop a biological weapon. It also provides an assessment of governability and a tailored set of recommendations that address security concerns. These recommendations are sensitive to the cost-benefit trade-off of regulating genome editing technologies. The book targets researchers as well as intelligence analysts, defense and security personnel, and policymakers.  

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Is CRISPR a Security Threat?

Recent advances in the CRISPR genome editing tool set

Emerging Life Sciences: New Challenges to Strategic Stability

• Gene Editing
• Biological Weapons
• synthetic biology

Table of contents (7 chapters)

Front matter, introduction: rapid technological advancements amid rising concerns of misuse.

Katherine Paris

Background: Genome Editing with Programmable Nucleases

Applying genetic engineering to biological weapons, risk of misuse assessment: part i, risk of misuse assessment: part ii, raising the alarm on crispr, conclusion: reducing the perils from the misuse of genome editing, back matter, authors and affiliations, about the author, bibliographic information.

Book Title : Genome Editing and Biological Weapons

Book Subtitle : Assessing the Risk of Misuse

Authors : Katherine Paris

DOI : https://doi.org/10.1007/978-3-031-21820-0

Publisher : Springer Cham

eBook Packages : Biomedical and Life Sciences , Biomedical and Life Sciences (R0)

Copyright Information : The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023

Hardcover ISBN : 978-3-031-21819-4 Published: 16 December 2022

Softcover ISBN : 978-3-031-21822-4 Published: 16 December 2023

eBook ISBN : 978-3-031-21820-0 Published: 15 December 2022

Edition Number : 1

Number of Pages : XXIV, 266

Topics : Genetics and Genomics , Biotechnology

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https://www.nist.gov/programs-projects/nist-genome-editing-program

NIST Genome Editing Program

Genome Editing technologies have transformed the potential of biosciences and biotechnology, by providing precision engineering tools that enable modifications to be made at specified positions within the genetic code of living cells. This rapidly evolving technology area is being adopted to advance many sectors of the bioeconomy including: human health (eg., cell and gene therapies, microbial-based diagnostics and therapeutics), agriculture, engineering/synthetic biology, environment, and biomanufacturing. The NIST Genome Editing Program develops standards, methods, tools, technology, and community norms to advance the reliability of genome editing technology and foster confidence in measurements for the genome editing field.

Vision: Foster technological innovation and enable quality in measurements to accelerate the translation and commercialization of  genome edited products

Goal: Develop measurement tools and standards to increase the confidence of utilizing genome editing technologies in research and commercial products

Description

For genome editing systems to reach their full potential in research and commercial products, new measurement tools, capabilities, and standards must be developed to efficiently implement and assess the performance of these editing technologies, as well as to evaluate the utility of resulting products (e.g., engineered cells) for their intended purposes.

The NIST Genome Editing Program actively supports this growing industry by:

• Evaluating measurement challenges related to implementing genome editing systems and understanding genome editing outcomes
• Qualifying analytical methods being used to detect and assess genome editing outcomes
• Developing new methods and standards to support confidence in detecting, interpreting, and reporting about genome editing outcomes

The NIST Genome Editing Program focus areas:

• Physical Measurements
• Data & Metadata
• Documentary Norms & Standards

NIST GENOME EDITING CONSORTIUM

The NIST Genome Editing Consortium is a public-private partnership with genome editing stakeholders to define measurement challenges for utilizing existing measurement capabilities to understand genome editing outcomes and develop shared solutions.

QUALIFICATION OF ASSAYS FOR DETECTING GENOME WIDE OFF-TARGET ACTIVITY OF GENOME EDITING MOLECULES

NIST is working closely with technology developers and other federal agencies to apply measurement assurance (including bioinformatics), associated tools, and well-documented protocols to improve reliability and reproducibility of recently developed assays for detecting off-target activity of genome editing molecules.

Project collaborators and assays currently under evaluation:

• NIH Somatic Cell Genome Editing (SCGE) Common Fund Program
• St. Jude Children’s Research Hospital, Lab of Shengdar Tsai, PhD
• DARPA SafeGenes Program
• Massachusetts General Hospital, Lab of J. Keith Joung, MD/PhD
• Health and Environmental Sciences Institute (HESI)
• AstraZeneca
• BrokenStrings Biosciences

ASSESSMENT OF GENOME EDITING REAGENTS & TECHNOLOGIES

Genome editing molecules to be introduced into a cell and/or organism can be formulated in various formats including: DNA, short RNA (in vitro transcribed and synthetic with or without modifications), mRNA, and protein. Additionally, there are several options for technologies to physically deliver genome editing molecules into cells and/or organisms. The NIST Genome Editing Program is actively assessing strategies for evaluating the properties, capabilities, and limitations of different genome editing molecule formulations as well as approaches for delivering genome editing molecules into cells.

Genome editing molecule delivery platforms under evaluation:

• FluidFM OMNIUM (Cytosurge AG)
• Neon (ThermoFisher)
• Nucleofector 4D (Lonza)

Any mention of commercial products within NIST web pages is for information only; it does not imply recommendation or endorsement by NIST.

PUBLICATIONS

• CHANGE-seq reveals genetic and epigenetic effects on CRISPR–Cas9 genome-wide activity
• The NIH Somatic Cell Genome Editing program
• Measurement and standardization challenges for extracellular vesicle therapeutic delivery vectors
• Fire Burn and Cauldron Bubble: What Is in Your Genome Editing Brew?
• Variability in genome-engineering source materials: consider your starting point

RELATED NEWS

• Spotlight on Commerce: Samantha Maragh, Ph.D., Leader of the Genome Editing Program, National Institute of Standards and Technology (NIST)
• Setting standards in the CRISPR genome editing revolution. Interview: Samantha Maragh (NIST)
• Rewritten in Our DNA: Measurements for Genome Editing
• 2021 GEORGE A. URIANO AWARD: Samantha Maragh

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Bacteria Encode Hidden Genes Outside Their Genome—Do We?

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Since the genetic code was first deciphered in the 1960s, our genes seemed like an open book. By reading and decoding our chromosomes as linear strings of letters, like sentences in a novel, we can identify the genes in our genome and learn why changes in a gene’s code affect health.

This linear rule of life was thought to govern all forms of life—from humans down to bacteria.

But a new study by Columbia researchers shows that bacteria break that rule and can create free-floating and ephemeral genes, raising the possibility that similar genes exist outside of our own genome.

“What this discovery upends is the notion that the chromosome has the complete set of instructions that cells use to produce proteins,” says Samuel Sternberg , associate professor of biochemistry & molecular biology at the Vagelos College of Physicians and Surgeons, who led the research with Stephen Tang, an MD/PhD student at the medical school.

“We now know that, at least in bacteria, there can be other instructions not preserved in the genome that are nonetheless essential for cell survival.”

“Astonishing” and “alien biology”

The scientific reaction had already made news a few months ago when the paper first appeared as a preprint. In a Nature News article, scientists called the discovery “alien biology,” “astonishing,” and “shocking.”

“It repeatedly left us in disbelief,” Tang says, “and we went from doubt to amazement as the mechanism gradually came into view.”

Bacteria and their viruses have been locked in battle for eons, as viruses try to inject their DNA into the bacterial genome and bacteria devise cunning methods (e.g. CRISPR) to defend themselves. Many bacterial defense mechanisms remain unexplored but could lead to new genome editing tools.

The bacterial defense system Sternberg and Tang picked to explore is an odd one: The system involves a piece of RNA with unknown function and a reverse transcriptase, an enzyme that synthesizes DNA from an RNA template. The most common defense systems in bacteria cut or degrade incoming viral DNA, “so we were puzzled by the idea of defending the genome by DNA synthesis,” Tang says.

Free-floating genes

To learn how the odd defense works, Tang first created a new technique to identify the DNA produced by the reverse transcriptase. The DNA he found was long but repetitive, containing multiple copies of a short sequence within the defense system’s RNA molecule.

Stephen Tang and Samuel Sternberg. Photo courtesy of the Sternberg lab at Columbia University Vagelos College of Physicians and Surgeons.

He then realized that this portion of the RNA molecule folds into a loop, and the reverse transcriptase travels numerous times around the loop to create the repetitive DNA. “It’s like you were intending to photocopy a book, but the copier just started churning out the same page over and over again,” Sternberg says.

The researchers originally thought something might be wrong with their experiments, or that the enzyme was making a mistake and the DNA it created was meaningless.

“This is when Stephen did some ingenious digging and found that the DNA molecule is a fully functioning, free-floating, transient gene,” Sternberg says.

The protein coded by this gene, the researchers found, is a critical part of the bacteria’s antiviral defense system. Viral infection triggers production of the protein (dubbed Neo by the researchers) which prevents the virus from replicating and infecting neighboring cells.

Extrachromosomal genes in humans?

If similar genes are found freely floating around in cells of higher organisms, “that would really be a game-changing discovery,” Sternberg says. “There might be genes, or DNA sequences, that don't reside in any of the 23 human chromosomes. Maybe they're only made in certain environments, in certain developmental or genetic contexts, and yet provide critical coding information that we rely on for our normal physiology.”

The lab is now using Tang’s methods to look for human extrachromosomal genes produced by reverse transcriptases.

Thousands of reverse transcriptase genes exist in the human genome and many have still undiscovered functions. “There is a significant gap to be filled that might reveal some more interesting biology,” Sternberg says.

Gene-editing wellspring

Though gene therapies that take advantage of CRISPR editing are in clinical trials (and one was approved last year for sickle cell), CRISPR is not the perfect technology.

New techniques that combine CRISPR with a reverse transcriptase are giving genome engineers more power. “The reverse transcriptase gives you the ability to write in new information at sites that CRISPR cuts, which CRISPR alone cannot do,” Tang says, “but everyone uses the same reverse transcriptase that was discovered decades ago.”

The reverse transcriptase that creates Neo has certain properties that may make it a better option for genome editing in the lab and for creating new gene therapies. And more mysterious reverse transcriptases exist in bacteria that are waiting to be explored.

“We think bacteria may have a treasure trove of reverse transcriptases that could be opportune starting points for new technologies once we understand how they work,” Sternberg says.

Additional information

All authors: Stephen Tang, Valentin Conte, Dennis J. Zhang, Rimantė Žedaveinytė, George D. Lampe, Tanner Wiegand, Lauren C. Tang, Megan Wang, Matt W.G. Walker, Jerrin Thomas George, Luke E. Berchowitz, Marko Jovanovic, and Samuel H. Sternberg.

The research was supported by the NIH (Medical Scientist Training Program grant T32GM145440, Ruth L. Kirchstein Individual Predoctoral Fellowship F30AI183830, R35GM124633, R01AG071869, and R01HG012216); the National Science Foundation (Graduate Research Fellowship and Award 2224211); a Human Frontier Science Program postdoctoral fellowship (LT001117/2021-C); the Schaefer Research Scholars Program; the Hirschl Family Trust, a Pew Biomedical Scholarship, an Irma T. Hirschl Career Scientist Award, start-up packages from Columbia University and the Columbia University Vagelos College of Physicians and Surgeons’ dean’s office; and the Vagelos Precision Medicine Fund.

Samuel Sternberg will be an investigator in the Howard Hughes Medical Institute beginning in autumn 2024.

Columbia University has filed a patent application related to this work. Samuel Sternberg is a co-founder and scientific advisor to Dahlia Biosciences, a scientific advisor to CrisprBits and Prime Medicine, and an equity holder in Dahlia Biosciences and CrisprBits.

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Caribou biosciences appoints tina albertson, md, phd, as chief medical officer.

-- Highly-experienced hematologist and oncologist with proven track record successfully driving global clinical development of CAR-T cell therapies --

BERKELEY, Calif., Aug. 12, 2024 (GLOBE NEWSWIRE) -- Caribou Biosciences, Inc.  (Nasdaq: CRBU), a leading clinical-stage CRISPR genome-editing biopharmaceutical company, today announced the appointment of Tina Albertson, MD, PhD, as chief medical officer. Dr. Albertson brings 15 years of experience leading clinical drug development of cellular therapies and biologics. She will be responsible for strategic leadership of the clinical, regulatory, and medical affairs functions, and provide medical and operational leadership of Caribou’s four clinical programs for hematologic malignancies and autoimmune diseases. Dr. Albertson will report to Rachel Haurwitz, PhD, Caribou’s president and chief executive officer.

Dr. Albertson was most recently the chief medical officer and head of development for Lyell Immunopharma, where she built and led the clinical development function. At Lyell, she initiated two Phase 1 clinical trials evaluating CAR-T cell and TIL therapies in solid tumors. Previously, Dr. Albertson was vice president of global drug development at Juno Therapeutics, a Bristol-Myers Squibb company, where she led the global development of BREYANZI (lisocabtagene maraleucel) from IND to filing of the initial BLA that resulted in FDA approval in large B cell lymphoma. At Juno, she led strategic development and execution of 9 global clinical trials, including 4 registrational trials of BREYANZI in other B cell malignancies and earlier lines of therapy. Dr. Albertson previously served as medical director of clinical development and experimental medicine at Seagen (formerly Seattle Genetics).

"Tina is an exceptional industry leader who brings significant experience in strategic clinical development of CAR-T cell therapies to Caribou. As a hematologist and oncologist, Tina has a deep understanding of the potential impact an off-the-shelf CAR-T cell therapy could have on patient treatment, outcomes, and reach,” said Dr. Haurwitz. “Her expertise in driving global clinical and regulatory strategies for cell therapies through all phases of development, including pivotal trials, will be valuable as we advance the development of our allogeneic CAR-T cell therapies in hematologic malignancies and autoimmune diseases.”

A photo accompanying this announcement is available at  https://www.globenewswire.com/NewsRoom/AttachmentNg/6816bea9-5ab8-4389-9bd6-e610f2c9e410

Dr. Albertson earned her MD from Stanford University and completed a clinical fellowship in pediatric hematology/oncology at Seattle Children’s Hospital and residency in pediatrics at Denver Children’s Hospital. She earned her PhD in cancer biology from University of Washington and her BS in molecular biology from the University of Oregon.

“Allogeneic CAR-T cell therapy holds immense promise as a transformative treatment modality, offering the potential to revolutionize the treatment landscapes for patients living with cancer or autoimmune disease,” said Dr. Albertson. “I am excited to join Caribou as the company is at the forefront of developing off-the-shelf CAR-T cell therapies and is working to deliver these promising treatment options to patients who desperately need them."

About Caribou’s novel next-generation CRISPR platform CRISPR genome editing uses easily designed, modular biological tools to make DNA changes in living cells. There are two basic components of Class 2 CRISPR systems: the nuclease protein that cuts DNA and the RNA molecule(s) that guide the nuclease to generate a site-specific, double-stranded break, leading to an edit at the targeted genomic site. CRISPR systems are capable of editing unintended genomic sites, known as off-target editing, which may lead to harmful effects on cellular function and phenotype. In response to this challenge, Caribou has developed CRISPR hybrid RNA-DNA guides (chRDNAs; pronounced “chardonnays”) that direct substantially more precise genome editing compared to all-RNA guides. Caribou is deploying the power of its Cas12a chRDNA technology to carry out high efficiency multiple edits, including multiplex gene insertions, to develop CRISPR-edited therapies.

About Caribou Biosciences, Inc. Caribou Biosciences is a clinical-stage CRISPR genome-editing biopharmaceutical company dedicated to developing transformative therapies for patients with devastating diseases. The company’s genome-editing platform, including its Cas12a chRDNA technology, enables superior precision to develop cell therapies that are armored to potentially improve activity against disease. Caribou is advancing a pipeline of off-the-shelf cell therapies from its CAR-T platform as readily available treatments for patients with hematologic malignancies and autoimmune diseases. Follow us @CaribouBio and visit www.cariboubio.com .

Forward-looking statements This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. In some cases, you can identify forward-looking statements by terms such as “may,” “will,” “should,” “expect,” “plan,” “anticipate,” “could,” “intend,” “target,” “project,” “contemplate,” “believe,” “estimate,” “predict,” “potential,” or “continue,” or the negative of these terms or other similar expressions, although not all forward-looking statements contain these words. These forward-looking statements include, without limitation, statements related to Caribou’s strategy, plans, and objectives, and expectations regarding its clinical and preclinical development programs. Management believes that these forward-looking statements are reasonable as and when made. However, such forward-looking statements are subject to risks and uncertainties, and actual results may differ materially from any future results expressed or implied by the forward-looking statements. Risks and uncertainties include, without limitation, risks inherent in the development of cell therapy products; uncertainties related to the initiation, cost, timing, progress, and results of Caribou’s current and future research and development programs, preclinical studies, and clinical trials; and the risk that initial, preliminary, or interim clinical trial data will not ultimately be predictive of the safety and efficacy of Caribou’s product candidates or that clinical outcomes may differ as patient enrollment continues and as more patient data becomes available; the risk that preclinical study results observed will not be borne out in human patients or different conclusions or considerations are reached once additional data have been received and fully evaluated; the ability to obtain key regulatory input and approvals; as well as other risk factors described from time to time in Caribou’s filings with the Securities and Exchange Commission, including its Annual Report on Form 10-K for the year ended December 31, 2023 and subsequent filings. In light of the significant uncertainties in these forward-looking statements, you should not rely upon forward-looking statements as predictions of future events. Except as required by law, Caribou undertakes no obligation to update publicly any forward-looking statements for any reason.

Caribou Biosciences, Inc. Contacts: Investors: Amy Figueroa, CFA [email protected]

Media: Peggy Vorwald, PhD [email protected]