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• U.S. Public Wary of Biomedical Technologies to ‘Enhance’ Human Abilities
• 2. U.S. public opinion on the future use of gene editing

Table of Contents

• 1. Understanding patterns in Americans’ reactions to gene editing, brain chip implants and synthetic blood transfusions that push boundaries of the human condition
• 3. Public opinion on the future use of brain implants
• 4. The public’s views on the future use of synthetic blood substitutes
• 5. From plastic surgery to vasectomies: Public opinion on current human enhancement options
• 6. Public sees science and technology as net positives for society
• Acknowledgments
• Methodology

Gene editing giving babies much reduced risk of serious disease

Respondents to the Pew Research Center survey read the following statement: “New developments in genetics and gene-editing techniques are making it possible to treat some diseases and conditions by modifying a person’s genes. In the future, gene-editing techniques could be used for any newborn, by changing the DNA of the embryo before it is born, and giving that baby a much reduced risk of serious diseases and conditions over his or her lifetime. Any changes to a baby’s genetic makeup could be passed on to future generations if they later have children, and over the long term this could change the genetic characteristics of the population.”

The potential genetic modification of humans and its ramifications have long been debated, but a recent scientific breakthrough in gene editing – a technique known as CRISPR – has raised the urgency of this conversation. In March and April of 2015, two separate groups of scientists published essays urging the scientific community to impose limits on genomic engineering, and the National Academies of Sciences, working in cooperation with the United Kingdom’s Royal Society and the Chinese Academy of Sciences, convened an international summit to discuss the science and policy of human gene editing. (For more details on these developments, see “ Human Enhancement: The Scientific and Ethical Dimensions of Striving for Perfection .”)

The Pew Research Center survey gauged, in broad terms, what the public thinks about the potential use of gene editing to enhance people’s health, in this case by reducing the probability of disease over a person’s lifetime. 11  Survey respondents were asked to consider a potential future scenario, republished in the accompanying sidebar, in which gene editing would be used to give healthy babies a much reduced risk of developing serious diseases. Gene-editing techniques are not currently being used in this way.

While many Americans say they would want to use such a technology for their own children, there is also considerable wariness when it comes to gene editing, especially among parents of minor children. Highly religious Americans are much more likely than those who are less religious to say they would not want to use gene-editing technology in their families. And, when asked about the possibility of using human embryos in the development of gene-editing techniques, a majority of adults – and two-thirds of those with high religious commitment– say this would make gene editing less acceptable to them.

This chapter focuses on these patterns and several others involving public attitudes about gene editing.

Despite some enthusiasm, the American public is largely wary about gene editing for babies

Americans have mixed emotional reactions to the possibility of using gene editing to reduce a baby’s risk of serious diseases, although more people express concern rather than enthusiasm. Fully two-thirds of U.S. adults (68%) say the prospect makes them either “very” or “somewhat” worried, while roughly half (49%) say they are “very” or “somewhat” enthusiastic about this technology. Three-in-ten adults are both enthusiastic and worried.

Asked to consider whether they would want this kind of gene editing for their own baby, Americans are split, with 48% saying they would want to use this technology for their child and a nearly identical share saying they would not. Parents who currently have a child under age 18 are less inclined than others to say they would want this kind of gene editing for their own baby; a clear majority of these parents (59%) would not want to use gene editing for their child.

Respondents also were asked whether they think “most people” would want to use gene-editing technology. Overall, a slim majority of Americans (55%) expect most people would want this kind of gene editing for their baby, while 42% say most people would not want this.

Those familiar with gene editing more inclined to want it for their own baby

Ideas about genetic modification and the potential for “designer babies” have been discussed among scientists, bioethicists and the broader public for some time.

When asked about their familiarity with gene editing, most Americans say they have heard either a little (48%) or a lot (9%) about this idea before, although a substantial minority (42%) had not heard anything about the possibility of gene editing before taking the survey.

Those who are at least somewhat familiar with the idea of gene editing are more inclined to say it is something they would want for their baby to reduce the child’s lifelong risk of certain serious diseases and conditions. Among those who have heard or read “a lot” or “a little” about gene-editing technology, 57% say they would want it for their child. But among those who had heard nothing at all prior to the survey, only 37% feel the same way. 12

Strong religious divides in preferences about gene editing

Personal preferences about gene editing are strongly tied to differences in religious commitment and affiliation.

Respondents were classified into high, medium and low levels of religious commitment based on the self-described importance of religion in their lives, frequency of worship service attendance and frequency of prayer. A person who says religion is very important in their life and who says they attend religious services at least weekly and pray at least daily is considered to have a “high” level of religious commitment, while a person who says religion is “not too” or “not at all” important in their life and who seldom or never attends religious services or prays is placed in the “low” commitment category. All others are classified as having “medium” commitment.

A majority of people with high religious commitment (64%) say they would not want gene editing for their own baby. By contrast, a nearly identical share of Americans with low religious commitment say they would want to use the technology for their child. Americans with a medium level of religious commitment are closely divided, with 48% saying they would want gene editing for their baby and 50% saying they would not.

There also are wide differences in feelings about gene editing by religious affiliation. White evangelical Protestants, who tend to be highly religious compared with other groups, are among the least likely to want their baby to have gene editing to reduce the risk of certain serious diseases (61% would not want it).

By contrast, majorities of atheists (75%) and agnostics (67%) would want to use gene editing for this purpose. Those who say their religion is “nothing in particular” are closely divided on this question, as are Catholics (both white and Hispanic) and white mainline Protestants.

Americans closely split on whether gene editing crosses a line that should not be crossed

The survey asked respondents whether the idea of editing genes to give healthy babies a much reduced risk of serious diseases and conditions is in keeping with other ways that humans have always tried to better themselves or whether “this idea is meddling with nature and crosses a line we should not cross.” Americans’ judgments on this question are closely divided, with 51% saying this idea is no different than other ways humans try to better themselves and 46% saying this idea crosses a line.

As with personal preferences for gene editing, there are wide differences on this issue by religious commitment. Fully 64% of those with a high level religious commitment say the idea of gene editing for healthy babies goes too far and is meddling with nature. By contrast, seven-in-ten of those with low religious commitment say this technology is no different from other things humans do to better themselves.

A majority of white evangelical Protestants (61%) consider the idea of gene editing for healthy babies to be crossing a line that should not be crossed. Black Protestants and Catholics are more divided over this question. Meanwhile, about eight-in-ten self-identified atheists (81%) and agnostics (80%) and roughly six-in-ten of those with no particular religious affiliation (58%) consider the idea of gene editing to be in keeping with other ways that humans try to better themselves.

Uncertainty, divisions over moral acceptability of gene editing

There is a large degree of uncertainty among Americans about whether gene editing is morally acceptable. A plurality of Americans (40%) say they are not sure whether it would be morally acceptable or not to edit a baby’s genes to give that child a reduced risk of developing serious diseases in their lifetime. Those who do express an opinion are evenly divided between those who consider gene editing for this purpose morally acceptable (28%) and those who consider it morally unacceptable (30%).

Among those with a view about this issue, there are wide differences by religious commitment. People with high religious commitment are more likely to say gene editing is morally unacceptable, while the balance of opinion leans in the opposite direction among those low in religious commitment.

‘Designer babies’ and views about genetic modification

A 2014 Pew Research Center survey asked people’s views about genetically modifying babies under two circumstances: in order to reduce a baby’s risk of serious diseases and conditions or to improve a baby’s intelligence. U.S. adults were closely split over whether it was “an appropriate use of medical advances” (46%) or “taking medical advances too far” (50%) to modify a baby’s genetic characteristics in order to reduce their risk of serious diseases. But, an overwhelming majority of adults (83%) said that modifying genetic characteristics to make a baby more intelligent was “taking medical advances too far.” Those who regularly attend worship services were more likely to consider genetic modifications for either purpose to be taking medical advances too far. Another 2014 Pew Research Center survey conducted with Smithsonian Magazine on public expectations for the future found two-thirds of Americans (66%) thought the possibility of parents being able to change the DNA of their children to produce smarter, healthier or more athletic offspring would be a change for the worse; 26% said this would be a change for the better.

Moral judgments about gene editing also vary by religious affiliation. Relatively few white evangelical Protestants and black Protestants say it is morally acceptable; just 16% and 15%, respectively. But a majority of atheists (60%) and half of agnostics (50%) say gene editing is morally acceptable.

Atheists and agnostics, meanwhile, are unlikely to call gene editing morally un acceptable; only about one-in-ten in each group say this is the case. By contrast, 43% of white evangelical Protestants say gene editing is morally unacceptable.

Still, substantial shares across all major religious groups – including roughly half of black Protestants and Hispanic Catholics – say they are not sure whether gene editing is morally acceptable.

To better understand people’s thinking about these issues, the Pew Research survey asked respondents to explain, in their own words, the reasons for their moral judgments about gene editing. The most common reasons mentioned by those with moral objections to gene editing referenced a belief that it would be altering “God’s plan” (34%) or that it would be going against nature or crossing a line we should not cross (26%), with some linking this idea to treating humans as a science experiment.

Other reasons Americans find gene editing to be morally unacceptable include the possibility of someone abusing the technology (9%); unintended consequences that may not be readily apparent until after implementation (8%); and the feeling that editing the genes of already-healthy people is unnatural or unnecessary (5%). Some 28% of those who say gene editing is morally unacceptable gave a different reason for feeling this way; an additional 27% did not give a reason.

Overall, 28% of U.S. adults say gene editing to give healthy babies a much reduced risk of serious diseases and conditions would be morally acceptable. The most common reasons for this point of view linked gene editing to other ways humans strive to improve themselves (32%), including some who framed this concept in terms of a moral responsibility for humans to use these tools if available and for parents to protect and improve a child’s health to the greatest extent possible. Another 21% mentioned improvements to society that would stem from gene editing, such as greater safety, health and productivity.

A plurality of adults –40% – is uncertain about the moral acceptability of gene editing for this purpose. While many of these respondents are simply unsure of their thinking or need more information on this issue, those who offered an explanation for their views were more likely to cite negative (53%) than positive (11%) effects of gene editing for society.

Public expects more negative than positive outcomes for society from gene editing

If gene editing is used to give healthy babies a reduced risk of serious diseases and conditions, Americans expect society to change. Nearly half of adults (46%) say such a development would change society “a great deal,” while 35% say it would change society “some” and just 17% say it would bring “not too much” or no change to society as whole.

A majority of U.S. adults expect the advent of gene editing could lead to widespread negative consequences for society. About three-quarters of adults (73%) say this technology would likely be used before the health effects are fully understood, and seven-in-ten say inequality would be prone to increase because gene editing would only be available for the wealthy.

A sizeable share of the public also sees the potential for positive outcomes, too, including about half who see increases in confidence for recipients of gene editing.

Acceptance of gene editing slightly higher for health effects that are less extreme

The survey asked a number of questions to tease out the way different possible extents of gene-editing technology would affect public thinking about the issue. In a hypothetical scenario in which the health effects of gene editing would make a person far healthier than any human known to date, Americans are more likely to say it would be taking the technology too far (54%) than to say it would be an appropriate use of technology (42%).

By comparison, people are more positive about gene editing when it has less-extreme health effects. In alternate scenarios in which gene editing would make a person always equally healthy to the average person today or much healthier than the average person today, Americans are somewhat more likely to see this as an appropriate use of technology than to say it is taking technology too far.

Greater acceptance of gene editing if there is control of its effects

Americans are more inclined to see gene editing that would give healthy babies a much reduced risk of serious diseases and conditions as acceptable under conditions that give those undergoing such procedures more control. For example, 41% of U.S. adults say gene editing would be more acceptable to them if people could choose which diseases and conditions are affected by the genetic modifications. By the same token, if the effects of gene editing would be permanent and irreversible, 37% of adults say gene editing would be less acceptable.

A key concern among bioethicists stems from the potential long-term implications of a type of gene editing that could change the human gene pool, known as germline editing. A person who undergoes germline editing would pass along their modified genes to any descendants; alternatively, gene editing done only in somatic cells would not be passed on to future offspring. Asked specifically about this possibility, people are more reluctant to embrace gene editing when it could affect future generations. Roughly half of adults (49%) say gene editing would be less acceptable to them if the effects “changed the genetic makeup of the whole population.” By contrast, about a third of Americans (34%) say they see an alternate scenario in which the effects of gene editing are limited to a single person as more acceptable.

The details of how gene editing is accomplished and assessed for this purpose are complex. According to experts, gene editing – whether for therapeutic purposes or enhancement – is likely to involve testing on human embryos. Indeed, the first research using CRISPR on human embryos was approved in the UK as of February 2016 . When survey respondents are asked to specifically consider the possibility that gene editing would involve testing on human embryos, most adults (54%) say this would make gene editing less acceptable to them.

The more religious Americans are, the more likely they are to oppose testing of gene-editing technology on human embryos. Fully two-thirds of highly religious adults say having to test the technology on human embryos would make gene editing less acceptable to them, compared with 42% of Americans with a low level of religious commitment.

When it comes to members of different religious groups, majorities of Protestants – including two-thirds of white evangelicals – and Catholics say gene editing that involved testing on human embryos would be less acceptable to them. Half of those with no particular religious affiliation (50%) also say testing on human embryos would make gene editing less acceptable.

After answering a number of questions about personal reactions to this idea and the likely effects for society of gene editing for this purpose, respondents were asked for an overall take on the expected effects on society as a whole of gene editing to give healthy babies a reduced risk of serious diseases. Slightly more Americans expect the benefits for society would outnumber the downsides of gene editing (36%). But some 28% say the downsides would outpace benefits, and a third (33%) say the downsides and benefits would even out.

Those with a high level of religious commitment are more likely to say the downsides would outnumber the benefits to society than they are to say the benefits would be more numerous (38% vs. 23%). But the opposite is true of those in the low religious commitment category; 46% say the benefits would outnumber the downsides, while 18% say there would be more downsides.

About six-in-ten atheists (59%) and roughly half of agnostics (53%) say the benefits of gene editing for this purpose would outnumber the downsides for society overall, while relatively few in these groups say the downsides would be greater. People with other religious identities are more divided on this question.

• A 2013 Pew Research Center report looked at public attitudes connected with aging and potential biomedical technologies to radically extend people’s lifespan. ↩
• The same pattern occurs among parents of minor children: 50% of those who have heard at least a little about this idea say they would definitely or probably want gene editing for their baby. This compares with 28% among parents who have not heard about this idea before. ↩

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• Published: 13 June 2024

Genome editing

RNA editing with CRISPR

• Petra Gross 1  

Nature Genetics volume  56 ,  page 1038 ( 2024 ) Cite this article

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• CRISPR-Cas9 genome editing
• CRISPR-Cas systems
• Genetic engineering
• Targeted gene repair

CRISPR-mediated selective DNA cleavage has revolutionized gene editing, but analogous methods to manipulate RNA, thus preserving the genome and avoiding potential adverse side effects, are not available. In their paper, Nemudraia et al. present an approach for the sequence-specific deletion of transcripts in human cells that makes use of type III-A CRISPR complexes, which cleave RNA in six-nucleotide increments. Type III-A CRISPR complexes have been previously used to knockdown RNA, but as shown here, the resulting CRISPR-guided RNA breaks are repaired by RtcB ligase in human cells, allowing for programmable RNA editing, for instance to restore gene function. As a proof-of-principle, the authors use this technology to remove premature stop codons in a reporter plasmid to restore protein expression in vitro, as well as excise clinically relevant nonsense mutations in the CFTR transcript in human bronchial epithelial cells carrying a disease allele. Furthermore, as type III CRISPR systems only require complementarity between the RNA guide and the RNA target, this method is highly adaptable and not restricted to the presence of any adjacent sequence motifs. Together, this work presents a versatile means to precisely edit RNA with potential therapeutic applications.

Original reference: Science 384 , 808–814 (2024)

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Gross, P. RNA editing with CRISPR. Nat Genet 56 , 1038 (2024). https://doi.org/10.1038/s41588-024-01816-5

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• There are 9 Let7 family members present in keratinocytes, all acting as translational suppressors of the same target mRNAs. This redundancy implies that Let7 is crucial to cellular functioning, and it has indeed been found to act as a tumor suppressor, down regulating functions such as proliferation, cell motility, and de-differentiation. I hypothesized that increasing loss of Let7 loci would lead to progressively more tumor-like phenotypes in the cells. Phenotypic analysis of these cells has not yet been possible, but initial Let7 knockouts were successful. The amyloid precursor protein is found on chromosome 21 and is strongly upregulated in individuals with Down Syndrome. This protein level is thought to play a role in the early onset development of amyloid-beta plaques in the brains of those with DS, one of the hallmarks of Alzheimer's Disease. APP was therefor chosen as a target for CRISPR, not to knock it out, but to delete one copy and reduce its gene dosage to normal levels. This would allow us to assess protein and RNA levels between trisomic and disomic APP, against a background of trisomy 21. If successful, our work will serve as a proof of principle for single knockout of any chromosome 21 gene. To attempt this, we need transient transfection, which is difficult to achieve with plasmids that can last in the cells for weeks. We hypothesized that brief periods of editing, and therefor incomplete knockouts, could be generated by transfecting cells with CRISPR RNAs.
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Let us assume that gene editing is safe—the role of safety arguments in the gene editing debate.

Published online by Cambridge University Press:  20 December 2018

This paper provides an analysis of the statement, made in many papers and reports on the use of gene editing in humans, that we should only use the technology when it is safe. It provides an analysis of what the statement means in the context of nonreproductive and reproductive gene editing and argues that the statement is inconsistent with the philosophical commitments of some of the authors, who put it forward in relation to reproductive uses of gene editing, specifically their commitment to Parfitian nonidentity considerations and to a legal principle of reproductive liberty.

But, if that is true it raises a question about why the statement is made. What is its discursive and rhetorical function? Five functions are suggested, some of which are more contentious and problematic than others. It is argued that it is possible, perhaps even likely, that the “only when it is safe” rider is part of a deliberate obfuscation aimed at hiding the full implications of the arguments made about the ethics of gene editing and their underlying philosophical justifications.

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Gene Editing, the Mystic Threat to Human Dignity

• Original Research
• Published: 18 March 2019
• Volume 16 , pages 249–257, ( 2019 )

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• Vera Lúcia Raposo   ORCID: orcid.org/0000-0001-7895-2181 1  

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Many arguments have been made against gene editing. This paper addresses the commonly invoked argument that gene editing violates human dignity and is ultimately a subversion of human nature. There are several drawbacks to this argument. Above all, the concept of what human dignity means is unclear. It is not possible to condemn a practice that violates human dignity if we do not know exactly what is being violated. The argument’s entire reasoning is thus undermined. Analyses of the arguments involved in this discussion have often led to the conclusion that gene editing contravenes the principle of genetic identity (genetic immutability) thereby subverting a requisite of human dignity and ultimately threatening human nature. This paper refutes these arguments and shows that any opposition to gene editing cannot rely on the human dignity argument.

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This work was supported by the University of Macau Multi-Year Research Grant MYRG2015-00007-FLL (Reproductive issues: juridical contextualization of reproductive techniques, genetics and new medical technologies. Some lessons from other legal orders).

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Raposo, V.L. Gene Editing, the Mystic Threat to Human Dignity. Bioethical Inquiry 16 , 249–257 (2019). https://doi.org/10.1007/s11673-019-09906-4

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Received : 27 April 2018

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Published : 18 March 2019

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DOI : https://doi.org/10.1007/s11673-019-09906-4

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AI plus gene editing promises to shift biotech into high gear

Professor of Chemistry, Connecticut College

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During her chemistry Nobel Prize lecture in 2018, Frances Arnold said, “Today we can for all practical purposes read, write and edit any sequence of DNA, but we cannot compose it.” That isn’t true anymore.

Since then, science and technology have progressed so much that artificial intelligence has learned to compose DNA, and with genetically modified bacteria, scientists are on their way to designing and making bespoke proteins.

The goal is that with AI’s designing talents and gene editing’s engineering abilities, scientists can modify bacteria to act as mini factories producing new proteins that can reduce greenhouse gases, digest plastics or act as species-specific pesticides.

As a chemistry professor and computational chemist who studies molecular science and environmental chemistry, I believe that advances in AI and gene editing make this a realistic possibility.

Gene sequencing – reading life’s recipes

All living things contain genetic materials – DNA and RNA – that provide the hereditary information needed to replicate themselves and make proteins. Proteins constitute 75% of human dry weight. They make up muscles, enzymes, hormones, blood, hair and cartilage. Understanding proteins means understanding much of biology. The order of nucleotide bases in DNA, or RNA in some viruses, encodes this information, and genomic sequencing technologies identify the order of these bases.

The Human Genome Project was an international effort that sequenced the entire human genome from 1990 to 2003. Thanks to rapidly improving technologies, it took seven years to sequence the first 1% of the genome and another seven years for the remaining 99%. By 2003, scientists had the complete sequence of the 3 billion nucleotide base pairs coding for 20,000 to 25,000 genes in the human genome.

However, understanding the functions of most proteins and correcting their malfunctions remained a challenge.

AI learns proteins

Each protein’s shape is critical to its function and is determined by the sequence of its amino acids, which is in turn determined by the gene’s nucleotide sequence. Misfolded proteins have the wrong shape and can cause illnesses such as neurodegenerative diseases, cystic fibrosis and Type 2 diabetes. Understanding these diseases and developing treatments requires knowledge of protein shapes.

Before 2016, the only way to determine the shape of a protein was through X-ray crystallography , a laboratory technique that uses the diffraction of X-rays by single crystals to determine the precise arrangement of atoms and molecules in three dimensions in a molecule. At that time, the structure of about 200,000 proteins had been determined by crystallography, costing billions of dollars.

AlphaFold, a machine learning program , used these crystal structures as a training set to determine the shape of the proteins from their nucleotide sequences. And in less than a year, the program calculated the protein structures of all 214 million genes that have been sequenced and published. The protein structures AlphaFold determined have all been released in a freely available database .

To effectively address noninfectious diseases and design new drugs, scientists need more detailed knowledge of how proteins, especially enzymes, bind small molecules. Enzymes are protein catalysts that enable and regulate biochemical reactions.

AlphaFold3 , released May 8, 2024, can predict protein shapes and the locations where small molecules can bind to these proteins. In rational drug design , drugs are designed to bind proteins involved in a pathway related to the disease being treated. The small molecule drugs bind to the protein binding site and modulate its activity, thereby influencing the disease path. By being able to predict protein binding sites, AlphaFold3 will enhance researchers’ drug development capabilities.

AI + CRISPR = composing new proteins

Around 2015, the development of CRISPR technology revolutionized gene editing. CRISPR can be used to find a specific part of a gene, change or delete it, make the cell express more or less of its gene product, or even add an utterly foreign gene in its place.

In 2020, Jennifer Doudna and Emmanuelle Charpentier received the Nobel Prize in chemistry “ for the development of a method (CRISPR) for genome editing .” With CRISPR, gene editing, which once took years and was species specific, costly and laborious, can now be done in days and for a fraction of the cost.

AI and genetic engineering are advancing rapidly. What was once complicated and expensive is now routine. Looking ahead, the dream is of bespoke proteins designed and produced by a combination of machine learning and CRISPR-modified bacteria. AI would design the proteins, and bacteria altered using CRISPR would produce the proteins. Enzymes produced this way could potentially breathe in carbon dioxide and methane while exhaling organic feedstocks, or break down plastics into substitutes for concrete.

I believe that these ambitions are not unrealistic, given that genetically modified organisms already account for 2% of the U.S. economy in agriculture and pharmaceuticals.

Two groups have made functioning enzymes from scratch that were designed by differing AI systems. David Baker ’s Institute for Protein Design at the University of Washington devised a new deep-learning-based protein design strategy it named “ family-wide hallucination ,” which they used to make a unique light-emitting enzyme . Meanwhile, biotech startup Profluent , has used an AI trained from the sum of all CRISPR-Cas knowledge to design new functioning genome editors .

If AI can learn to make new CRISPR systems as well as bioluminescent enzymes that work and have never been seen on Earth, there is hope that pairing CRISPR with AI can be used to design other new bespoke enzymes. Although the CRISPR-AI combination is still in its infancy, once it matures it is likely to be highly beneficial and could even help the world tackle climate change.

It’s important to remember, however, that the more powerful a technology is, the greater the risks it poses. Also, humans have not been very successful at engineering nature due to the complexity and interconnectedness of natural systems, which often leads to unintended consequences.

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It could cure the incurable, revolutionize vaccines and immortalize cells: RNA explained

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Ask a friend what DNA is and, chances are, they have a general idea.

Seventy years after scientists discovered the two-stranded helix, DNA (deoxyribonucleic acid) is widely understood as the keeper of our genetic information and a window into our ancestry. It has become a household word.

Not so with RNA.

Book cover of “The Catalyst: RNA and the Quest to Unlock Life’s Deepest Secrets” by Tom Cech, winner of the Nobel Prize

“RNA was never the star of the show,” writes Tom Cech, a distinguished professor of biochemistry at CU Boulder who won the Nobel Prize in Chemistry in 1989 for his research on RNA. “It was like a biochemical backup singer slaving away in the shadows of the diva.”

As Cech reveals in his new book “The Catalyst: RNA and the Quest to Unlock Life’s Deepest Secrets,” RNA (ribonucleic acid) is having its moment, with research surging globally and more than 400 RNA-based drugs in development.

One variety, known as messenger RNA, could ultimately lead to a one-shot immunization that renders seasonal flu shots obsolete. Another guides CRISPR, a molecular set of scissors that can edit out mutations in the genetic code to prevent or cure deadly diseases. An RNA-powered enzyme called telomerase can also—for better or worse—forestall aging in human cells, a tantalizing but misunderstood idea for those seeking the Fountain of Youth.

CU Boulder Today sat down with Cech to discuss CU Boulder’s rich heritage of RNA research and the oft-overlooked molecule he has dedicated his career to.

What prompted you to write this book?

During the pandemic, my lab was shut down but my subject was suddenly on the tip of everyone’s tongue. People started hearing more about what RNA was and what messenger RNA might be able to do. Some were excited; some were hesitant. I thought that more information is always a good thing, so I decided to reach out to the non-scientific public and try to explain it.

In a nutshell, what is RNA?

RNA is a copy of one of the two strands in DNA. It is best known as a messenger that carries the information from DNA out of the cell nucleus to orchestrate the synthesis of proteins. It is tiny: If you stacked molecules of RNA side by side, you could fit 50,000 of them within the breadth of a single human hair. What it lacks in size it makes up for in versatility. Because it is a single strand, RNA can fold itself in myriad different ways that give it a huge list of roles beyond just being a messenger.

What did your Nobel-winning team discover in 1989?

We revealed that RNA could also be a catalyst. That means it could cut and join biochemical bonds, speeding up reactions necessary for life to exist. There are dozens of other equally thrilling things that RNA is capable of. But it was one of the moments in science when people woke up to thinking they had underestimated RNA, and they should keep their eyes open for new things it could do.

They found them. Since 2000, RNA-related breakthroughs have led to 11 Nobel prizes (including the 2020 prize to Jennifer Doudna , who did her postdoctoral work at CU Boulder, for the co-development of CRISPR).

What’s the connection between COVID and RNA?

The coronavirus itself is an RNA virus. It doesn’t have a genome made of DNA like we do. Instead, it uses RNA both to store its genetic information (yes RNA can do that, too) and as a messenger to make the proteins it needs to continue its infectious cycle. Ebola, polio and influenza are also RNA viruses.

What was so revolutionary about the ‘mRNA’ COVID vaccines?

To vaccinate against a virus, we typically inject a person with a disabled form of a virus. That can be a little frightening to think about. mRNA vaccines take that concern away because they are not made of virus. Instead, they are made of messenger RNA that instructs the body itself to make a protein—the spike protein in the case of COVID.

It gives the immune system a heads up and says, “If you ever see anything that looks like this, it's gonna be bad, so you need to mount a cellular response to kill it.”

Cech, left, with Jennifer Doudna, who won the Nobel Prize in chemistry in 2020 for co-designing the RNA-guided gene-editing tool CRISPR.

Could this improve vaccines in general?

That is the hope. For instance, believe it or not, we currently inject about a million chicken eggs annually with the flu virus to make the seasonal flu vaccine. It takes so long that we have to guess what strain will be prominent during the next flu season and sometimes we are wrong. That’s why it’s only between 30% and 60% efficacious.

With mRNA vaccines, the process is so simple, someone could design the vaccine in about a week. The hope is that vaccine manufacturers could wait until they know what strain was going around and then design a much more effective vaccine or design a one-and-done and the seasonal shot would become a thing of the past.

Telomerase has become a darling of the biotech and supplement industries. Is it really an anti-aging miracle?

Chromosomes are like little strings of DNA pearls. In the absence of telomerase, the pearl at the end is lost each time a cell divides, and the string becomes shorter and shorter. Telomerase is a collaboration between RNA and a protein called telomerase reverse transcriptase, which was discovered at CU Boulder. It continually adds pearls to the end of the necklace (known as the telomere) rendering cells forever young.

Multiple diseases have been traced to low levels of telomerase. These individuals would greatly benefit from a way to lengthen the telomeres of their stem cells. But on the other hand, what is a tumor cell? Well, it’s immortal. So an anti-telomerase therapy could be useful in treating cancer.

The fact that telomerase can immortalize human cells is a scientific fact, but to suggest that an increase in the level of telomerase could extend human life span generally is overly simplistic.

Can you provide a few examples of RNA-based medications in use today?

Spinal muscular atrophy (SMA) is a reasonably common and deadly childhood disease that has been treated successfully with an RNA therapy. The first CRISPR therapy was approved late last year for sickle cell disease. CU Boulder scientists have developed RNA-based therapies for macular degeneration, and some are trying to tackle Alzheimer’s disease.

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Right gene in the right place —

Iv infusion enables editing of the cystic fibrosis gene in lung stem cells, approach relies on lipid capsules like those in the mrna vaccines..

John Timmer - Jun 13, 2024 9:53 pm UTC

The development of gene editing tools, which enable the specific targeting and correction of mutations, hold the promise of allowing us to correct those mutations that cause genetic diseases. However, the technology has been around for a while now—two researchers were critical to its development in 2020— and there have been only a few cases where gene editing has been used to target diseases.

One of the reasons for that is the challenge of targeting specific cells in a living organism. Many genetic diseases affect only a specific cell type, such as red blood cells in sickle-cell anemia, or specific tissue. Ideally, to limit potential side effects, we'd like to ensure that enough of the editing takes place in the affected tissue to have an impact, while minimizing editing elsewhere to limit side effects. But our ability to do so has been limited. Plus, a lot of the cells affected by genetic diseases are mature and have stopped dividing. So, we either need to repeat the gene editing treatments indefinitely or find a way to target the stem cell population that produces the mature cells.

On Thursday, a US-based research team said that they've done gene editing experiments that targeted a high-profile genetic disease: cystic fibrosis. Their technique largely targets the tissue most affected by the disease (the lung), and occurs in the stem cell populations that produce mature lung cells, ensuring that the effect is stable.

Getting specific

The foundation of the new work is the technology that gets the mRNAs of the COVID-19 mRNA vaccines inside cells. The nucleic acids of an mRNA are large molecules with a lot of charged pieces, which makes it difficult for them to cross a membrane to get inside of a cell. To overcome that problem, the researchers package the mRNA inside a bubble of lipids, which can then fuse with cell membranes, dumping the mRNA inside the cell.

This process, as the researchers note, has two very large advantages: We know it works, and we know it's safe. "More than a billion doses of lipid nanoparticle–mRNA COVID-19 vaccines have been administered intramuscularly worldwide," they write, "demonstrating high safety and efficacy sustained through repeatable dosing." (As an aside, it's interesting to contrast the research community's view of the mRNA vaccines to the conspiracies that circulate widely among the public.)

There's one big factor that doesn't matter for vaccine delivery but does matter for gene editing: They're not especially fussy about what cells they target for delivery. So, if you want to target something like blood stem cells, then you need to alter the lipid particles in some way to get them to preferentially target the cells of your choice.

There are a lot of ideas on how to do this, but the team behind this new work found a relatively simple one: changing the amount of positively charged lipids on the particle. In 2020, they published a paper in which they describe the development of selective organ targeting (SORT) lipid nanoparticles. By default, many of the lipid particles end up in the liver. But, as the fraction of positively charged lipids increases, the targeting shifts to the spleen and then to the lung.

So, presumably, because they know they can target the lung, they decided to use SORT particles to send a gene editing system specific to cystic fibrosis, which primarily affects that tissue and is caused by mutations in a single gene. While it's relatively easy to get things into the lung, it's tough to get them to lung cells, given all the mucus, cilia, and immune cells that are meant to take care of foreign items in the lung.

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Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing

Misganaw asmamaw.

1 Division of Biochemistry, Department of Biomedical Sciences, College of Medicine and Health Sciences, Debre Tabor University, Debre Tabor, Ethiopia

Belay Zawdie

2 Division of Biochemistry, Department of Biomedical Sciences, Institute of Health, Jimma University, Jimma, Ethiopia

Clustered regularly interspaced short palindromic repeat (CRISPR) and their associated protein (Cas-9) is the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The mechanism of CRISPR/Cas-9 genome editing contains three steps, recognition, cleavage, and repair. The designed sgRNA recognizes the target sequence in the gene of interest through a complementary base pair. While the Cas-9 nuclease makes double-stranded breaks at a site 3 base pair upstream to protospacer adjacent motif, then the double-stranded break is repaired by either non-homologous end joining or homology-directed repair cellular mechanisms. The CRISPR/Cas-9 genome-editing tool has a wide number of applications in many areas including medicine, agriculture, and biotechnology. In agriculture, it could help in the design of new grains to improve their nutritional value. In medicine, it is being investigated for cancers, HIV, and gene therapy such as sickle cell disease, cystic fibrosis, and Duchenne muscular dystrophy. The technology is also being utilized in the regulation of specific genes through the advanced modification of Cas-9 protein. However, immunogenicity, effective delivery systems, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. Although CRISPR/Cas-9 becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications, there are still challenges to rub in the practical applications and various improvements are needed to overcome obstacles.

Genome editing is a type of genetic engineering in which DNA is deliberately inserted, removed, or modified in living cells. 1 The name CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) refers to the unique organization of short, partially repeated DNA sequences found in the genomes of prokaryotes. CRISPR and its associated protein (Cas-9) is a method of adaptive immunity in prokaryotes to defend themselves against viruses or bacteriophages. 2 Japanese scientist Ishino and his team accidentally found unusual repetitive palindromic DNA sequences interrupted by spacers in Escherichia coli while analyzing a gene for alkaline phosphatase first discovered CRISPR in 1987. However, they did not ascertain its biological function. In 1990, Francisco Mojica identifies similar sequences in other prokaryotes and he named CRISPR, yet the functions of these sequences were a mystery. 3 Later on in 2007, a CRISPR was experimentally conferred as a key element in the adaptive immune system of prokaryotes against viruses. During the adaptation process, bacterial cells become immunized by the insertion of short fragments of viral DNA (spacers) into a genomic region called the CRISPR array. Hence, spacers serve as a genetic memory of previous viral infections. 4 The CRISPR defense mechanism protects bacteria from repeated viral attacks via three basic stages: adaptation (spacer acquisition), crRNA synthesis (expression), and target interference. CRISPR loci are an array of short repeated sequences found in chromosomal or plasmid DNA of prokaryotes. Cas gene is usually found adjacent to CRISPR that codes for nuclease protein (Cas protein) responsible to destroy or cleave viral nucleic acid. 5

Before the discovery of CRISPR/Cas-9, scientists were relied on two gene-editing techniques using restriction enzymes, zinc finger nucleases (ZFN) and Transcription activator-like effector nucleases (TALENs). 6 ZFN has a zinc finger DNA binding domain used to bind a specific target DNA sequence and a restriction endonuclease domain used to cleave the DNA at the target site. TALENs are also composed of DNA binding domain and restriction domain like ZFN but their DNA binding domain has more potential target sequence than the ZFN gene-editing tool. In both cases, the difficulty of protein engineering, being expensive, and time-consuming were the major challenges for researchers and manufacturers. 6 , 7 The development of a reliable and efficient method of a gene-editing tool in living cells has been a long-standing goal for biomedical researchers. After figuring out the CRISPR mechanism in prokaryotes, scientists understood that it could have beneficial use in humans, plants, and other microbes. It was in 2012 that Doudna, J, and Charpentier, E discovered CRISPR/Cas-9 could be used to edit any desired DNA by just providing the right template. 8 Since then, CRISPR/Cas-9 becomes the most effective, efficient, and accurate method of genome editing tool in all living cells and utilized in many applied disciplines. 9 Thus, this review aims to discuss the mechanisms of genome editing mediated by CRISPR/Cas-9 and to highlight its recent applications as one of the most important scientific discoveries of this century, as well as the current barriers to the transformation of this technology.

Components of CRISPR/Cas-9

Based on the structure and functions of Cas-proteins, CRISPR/Cas system can be divided into Class I (type I, III, and IV) and Class II (type II, V, and VI). The class I systems consist of multi-subunit Cas-protein complexes, while the class II systems utilize a single Cas-protein. Since the structure of type II CRISPR/Cas-9 is relatively simple, it has been well studied and extensively used in genetic engineering. 10 Guide RNA (gRNA) and CRISPR-associated (Cas-9) proteins are the two essential components in CRISPR/Cas-9 system. The Cas-9 protein, the first Cas protein used in genome editing was extracted from Streptococcus pyogenes (SpCas-9). It is a large (1368 amino acids) multi-domain DNA endonuclease responsible for cleaving the target DNA to form a double-stranded break and is called a genetic scissor. 11 Cas-9 consists of two regions, called the recognition (REC) lobe and the nuclease (NUC) lobe. The REC lobe consists of REC1 and REC2 domains responsible for binding guide RNA, whereas the NUC lobe is composed of RuvC, HNH, and Protospacer Adjacent Motif (PAM) interacting domains. The RuvC and HNH domains are used to cut each single-stranded DNA, while PAM interacting domain confers PAM specificity and is responsible for initiating binding to target DNA. 12 Guide RNA is made up of two parts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). The crRNA is an 18–20 base pair in length that specifies the target DNA by pairing with the target sequence, whereas tracrRNA is a long stretch of loops that serve as a binding scaffold for Cas-9 nuclease. In prokaryotes, the guide RNA is used to target viral DNA, but in the gene-editing tool, it can be synthetically designed by combining crRNA and tracrRNA to form a single guide RNA (sgRNA) in order to target almost any gene sequence supposed to be edited. 11

Mechanisms of CRISPR/CAS-9 Genome Editing

The mechanism of CRISPR/Cas-9 genome editing can be generally divided into three steps: recognition, cleavage, and repair. 13 The designed sgRNA directs Cas-9 and recognizes the target sequence in the gene of interest through its 5ʹcrRNA complementary base pair component. The Cas-9 protein remains inactive in the absence of sgRNA. The Cas-9 nuclease makes double-stranded breaks (DSBs) at a site 3 base pair upstream to PAM. 14 PAM sequence is a short (2–5 base-pair length) conserved DNA sequence downstream to the cut site and its size varies depending on the bacterial species. The most commonly used nuclease in the genome-editing tool, Cas-9 protein recognizes the PAM sequence at 5ʹ-NGG-3ʹ (N can be any nucleotide base). Once Cas-9 has found a target site with the appropriate PAM, it triggers local DNA melting followed by the formation of RNA-DNA hybrid, but the mechanism of how Cas-9 enzyme melts target DNA sequence was not clearly understood yet. Then, the Cas-9 protein is activated for DNA cleavage. HNH domain cleaves the complementary strand, while the RuvC domain cleaves the non-complementary strand of target DNA to produce predominantly blunt-ended DSBs. Finally, the DSB is repaired by the host cellular machinery. 11 , 15

Double-Stranded Break Repair Mechanisms

Non-homologous end joining (NHEJ), and homology-directed repair (HDR) pathways are the two mechanisms to repair DSBs created by Cas-9 protein in CRISPR/Cas-9 mechanism. 16 NHEJ facilitates the repair of DSBs by joining DNA fragments through an enzymatic process in the absence of exogenous homologous DNA and is active in all phases of the cell cycle. It is the predominant and efficient cellular repair mechanism that is most active in the cells, but it is an error-prone mechanism that may result in small random insertion or deletion (indels) at the cleavage site leading to the generation of frameshift mutation or premature stop codon. 17 HDR is highly precise and requires the use of a homologous DNA template. It is most active in the late S and G2 phases of the cell cycle. In CRISPR-gene editing, HDR requires a large amount of donor (exogenous) DNA templates containing a sequence of interest. HDR executes the precise gene insertion or replacement by adding a donor DNA template with sequence homology at the predicted DSB site. 16 , 17

Applications of CRISPR/CAS-9

In just a few years of its discovery, the CRISPR/Cas-9 genome editing tool has already being explored for a wide number of applications and had a massive impact on the world in many areas including medicine, agriculture, and biotechnology. In the future, researchers hope that this technology will continue to advance for treating and curing diseases, develop more nutritious crops, and eradicating infectious diseases. 18 Highlights for some of the recent CRISPR/Cas-9 applications and clinical trials being investigated are discussed below.

Role in Gene Therapy

More than 6000 genetic disorders have been known so far. But the majority of the diseases lack effective treatment strategies. 19 Gene therapy is the process of replacing the defective gene with exogenous DNA and editing the mutated gene at its native location. It is the latest development in the revolution of medical biotechnology. From 1998 to August 2019, 22 gene therapies including the novel CRISPR/Cas-9 have been approved for the treatment of human diseases. 20

Since its discovery in 2012, CRISPR/Cas-9 gene editing has held the promise of curing most of the known genetic diseases such as sickle cell disease, β-thalassemia, cystic fibrosis, and muscular dystrophy. 21 , 22 CRISPR/Cas-9 for targeted sickle cell disease (SCD) therapy and β-thalassemia have been also applied in clinical trials. 23 SCD is an autosomal recessive genetic disease of red blood cells, which occurs due to point mutation in the β-globin chain of hemoglobin leading to sickle hemoglobin (HbS). During the deoxygenation process, HbS polymerization leads to severe clinical complications like hemolytic anemia. 24 Either direct repairing the gene of hemoglobin S or boosting fetal γ-globin are the two main approaches that CRISPR/Cas-9 is being used to treat SCD. 25 However, the most common method used in a clinical trial is based on the approach of boosting fetal hemoglobin. First bone marrow cells are removed from patients and the gene that turns off fetal hemoglobin production, called B-cell Lymphoma 11A (BCL11A) is disabled with CRISPR/Cas-9. Then, the gene-edited cells are infused back into the body. 26 BCL11A is a 200 base pair gene found on chromosome 2 and its product is responsible to switch γ-globin into the β-globin chain by repressing γ-globin gene expression. 27 Once this gene is disabled using CRISPR/Cas-9, the production of fetal hemoglobin containing γ-globin in the red blood cells will increase, thereby alleviating the severity and manifestations of SCD. 28

Scientists have been also investigating CRISPR/Cas-9 for the treatment of cystic fibrosis. The genetic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene decreases the structural stability and function of CFTR protein leading to cystic fibrosis. 29 CFTR protein is an anion channel protein regulated by protein kinase-A, located at the apical surface of epithelial cells of the lung, intestine, pancreas, and reproductive tract. 30 Although there is no cure for cystic fibrosis, symptom-based therapies (such as antibiotics, bronchodilators, and mucus thinning medications) and CFTR modulating drugs have become the first-line treatments to relieve symptoms and reduce the risk of complications. 31 Currently, gene manipulation technologies and molecular targets are also being explored. The use of CRISPR/Cas-9 technology for genome editing has great potential, although it is in the early stages of development. 32 In 2013, researchers culture intestinal stem cells from two cystic fibrosis patients and corrected the mutation at the CFTR locus resulting in the expression of the correct gene and full function of the protein. Since then, the potential utility of the application of CRISPR/Cas-9 for cystic fibrosis was established. 33 Furthermore, Duchenne muscular dystrophy (DMD), which is caused by a mutation in the dystrophin gene and characterized by muscle weakness, has been successfully corrected by CRISPR/Cas-9 in patient-induced pluripotent stem cells. 34 Despite considerable efforts, the treatment available for DMD remains supportive rather than curative. Currently, several therapeutic approaches (gene therapy, cell therapy, and exon skipping) have been investigated to restore the expression of dystrophin in DMD muscles. 35 , 36 Deletion/excision of intragenic DNA and removing the duplicated exon by CRISPR/Cas-9 are the new and promising approaches in correcting the DMD gene, which restores the expression of dystrophin protein. 37

Moreover, the latest researches show that the CRISPR/Cas-mediated single-base editing and prime editing systems can directly install mutations in cellular DNA without the need for a donor template. The CRISPR/Cas-base editor and prime editor system do not produce DSB, which reduces the possibility of indels that are different from conventional Cas-9. 38 So far, two types of base editors have been developed: cytosine base editor (CBE) and adenine base editor (ABE). 39 The CBE is a type of base editor composed of cytidine deaminase fused with catalytically deficient or dead Cas-9 (dCas-9). It is one of the novel gene therapy strategies that can produce precise base changes from cytidine (C) to thymidine (T). 40 However, the target range of the CBE base editor is still restricted by PAM sequences containing G, T, or A bases. Recently, a more advanced fidelity and efficiency base editor called nNme2-CBE (discovered from Neisseria meningitides ) with expanded PAM compatibility for cytidine dinucleotide has been developed in both human cells and rabbits embryos. 41 The ABE uses adenosine deaminase fused to dCas-9 to correct the base-pair change from adenosine (A) to guanosine (G). 38 Overall, single-base editing through the fusion of dCas-9 to cytidine deaminase or adenosine deaminase is a safe and efficient method to edit point mutations. But both base editors can only fix four-transition mutations (purine to purine or pyrimidine to pyrimidine). 42 To overcome this shortcoming, the most recent member of the CRISPR genome editing toolkit called Prime Editor (PE) has been developed to extend the scope of DNA editing beyond the four types of transition mutations. 43 PE contains Cas-9 nickase fused with engineered reverse transcriptase and multifunctional primer editing guide RNA (pegRNA). The pegRNA recognizes the target nucleotide sequence; the Cas-9 nickase cuts the non-complementary strand of DNA three bases upstream from the PAM site, exposing a 3ʹ-OH nick of genomic DNA. The reverse transcriptase then extends the 3ʹ nick by copying the edit sequence of pegRNA. Hence, PE not only corrects all 12 possible base-to-base transitions, and transversion mutations but also small insertion and deletion mutations in genetic disorders. 44

Therapeutic Role of CRISPR/Cas-9

The first CRISPR-based therapy in the human trial was conducted to treat patients with refractory lung cancer. Researchers first extract T-cells from three patient’s blood and they engineered them in the lab through CRISPR/Cas-9 to delete genes ( TRAC , TRBC , and PD-1 ) that would interfere to fight cancer cells. Then, they infused the modified T-cells back into the patients. The modified T-cells can target specific antigens and kill cancer cells. Finally, no side effects were observed and engineered T-cells can be detected up to 9 months of post-infusion. 45 CRISPR/Cas-9 gene-editing technology could also be used to treat infectious diseases caused by microorganisms. 46 One focus area for the researchers is treating HIV, the virus that leads to AIDS. In May 2017, a team of researchers from Temple University demonstrated that HIV-1 replication can be completely shut down and the virus eliminated from infected cells through excision of HIV-1 genome using CRISPR/Cas-9 in animal models. 47 In addition to the approach of targeting the HIV-genome, CRISPR/Cas-9 technology can also be used to block HIV entry into host cells by editing chemokine co-receptor type-5 ( CCR5 ) genes in the host cells. For instance, an in vitro trial conducted in China reported that genome editing of CCR5 by CRISPR/Cas-9 showed no evidence of toxicity (infection) on cells and they concluded that edited cells could effectively be protected from HIV infection than unmodified cells. 48

Role in Agriculture

As the world population continues to grow, the risk of shortage in agricultural resources is real. Hence, there is a need for new technologies for increasing and improving natural food production. CRISPR/Cas-9 is an existing addition to the field since it has been used to genetically modify foods to improve their nutritional value, increase their shelf life, make them drought-tolerant, and enhance disease resistance. 18 There are generally three ways that CRISPR is solving the world’s food crisis. It can restore food supplies, help plants to survive in hostile conditions, and could improve the overall health of the plants. 49

Role in Gene Activation and Silencing

Beyond genome editing activity, CRISPR/Cas-9 can be used to artificially regulate (activate or repress) a certain target of a gene through advanced modification of Cas-9 protein. 15 Researchers had performed an advanced modified Cas-9 endonuclease called dCas-9 nuclease by inactivating its HNH and RuvC domains. The dCas-9 nuclease lacks DNA cleavage activity, but its DNA binding activity is not affected. Then, transcriptional activators or inhibitors can be fused with dCas-9 to form the CRISPR/dCas-9 complex. Therefore, catalytically inactive dCas-9 can be used to activate (CRISPRa) or silence (CRISPRi) the expression of a specific gene of interest. 50 Moreover, the CRISPR/dCas-9 can be also used to visualize and pinpoint where specifically the gene of interest is located inside the cell (subcellular localization) by fusing a marker such as Green Fluorescent Proteins (GFP) with dCas-9 enzyme. This enables site-specific labeling and imaging of endogenous loci in living cells for further utilization. 51

Challenges for CRISPR/Cas-9 Application

Despite its great promise as a genome-editing system CRISPR/Cas-9 technology had hampered by several challenges that should be addressed during the process of application. Immunogenicity, lack of a safe and efficient delivery system to the target, off-target effect, and ethical issues have been the major barriers to extend the technology in clinical applications. 52 Since the components of the CRISPR/Cas-9 system are derived from bacteria, host immunity can elicit an immune response against these components. Researchers also found that there were both pre-existing humoral (anti-Cas-9 antibody) and cellular (anti-Cas-9 T cells) immune responses to Cas-9 protein in healthy humans. Therefore, how to detect and reduce the immunogenicity of Cas-9 protein is still one of the most important challenges in the clinical trial of the system. 53

Safe and effective delivery of the components into the cell is essential in CRISPR/Cas-9 gene editing. Currently, there are three methods of delivering the CRISPR/Cas-9 complex into cells, physical, chemical, and viral vectors. Non-viral (physical and chemical) methods are more suitable for ex vivo CRISPR/Cas-9-based gene editing therapy. 54 The physical methods of delivering CRISPR/Cas-9 can include electroporation, microinjection, hydrodynamic injection, and so on. Electroporation applies a strong electric field to the cell membrane to temporarily increase the permeability of the membrane, allowing the CRISPR/Cas-9 complex to enter the cytoplasm of the target cell. However, the main limitation of this method is that it causes significant cell death. 55 Microinjection involves injecting the CRISPR/Cas-9 complex directly into cells at the microscopic level for rapid gene editing of a single cell. Nevertheless, this method also has several disadvantages such as cell damage, which is technically challenging and is only suitable for a limited number of cells. 56 The hydrodynamic injection is the rapid injection of a large amount of high-pressure liquid into the bloodstream of animals, usually using the tail vein of mice. Although this method is simple, fast, efficient, and versatile, it has not yet been used in clinical applications due to possible complications. 57 The chemical methods of CRISPR/Cas-9 delivery involves lipid and polymer-based nanoparticles. 58 Lipid nanoparticles/liposomes are spherical structures composed of lipid bilayers membrane and are synthesized in aqueous solutions using Lipofectamine-based reagents. The positively charged liposomes encapsulated with negatively charged nucleic acids thereby facilitate the fusion of the complex across the cell membrane into cells. 59 Polymeric nanoparticles, such as polyethyleneimine and poly-L-lysine, are the most widely used carriers of CRISPR/Cas-9 components. Like lipid nanoparticles, polymer-based nanoparticles can also transverse the complex in the membrane through endocytosis. 60

Viral vectors are the natural experts for in vivo CRISPR/Cas-9 delivery. 61 Vectors, such as adenoviral vectors (AVs), adeno-associated viruses (AAVs), and lentivirus vectors (LVs) are currently being widely used as delivery methods due to their higher delivery efficiency relative to physical and chemical methods. Among them, AAVs are the most commonly used vectors due to their low immunogenicity and non-integration into the host cell genome compared to other viral vectors. 62 However, the limited virus cloning capacity and the large size of the Cas-9 protein remain a major problem. One strategy to tackle this hurdle is to package sgRNA and Cas-9 into separate AAVs and then co-transfect them into cells. Recent methods employ a smaller strain of Cas-9 from Staphylococcus aureus (SaCas-9) instead of the more commonly used SpCas-9 to allow packaging of sgRNA and Cas-9 in the same AAVs. 54 , 61 Lately, the development of extracellular vesicles (EVs), for the in vivo delivery of CRISPR/Cas-9 to avoid some of the limitations of viral and non-viral methods has shown a great potential. 63

The designed sgRNA will mismatch to the non-target DNA and can result in nonspecific, unexpected genetic modification, which is called the off-target effect. 57 The CRISPR/Cas-9 target efficiency is determined by the 20-nucleotide sequences of sgRNA and the PAM sequences adjacent to the target genome. It has been shown that more than three mismatches between the target sequence and the 20-nucleotide sgRNA can result in off-target effects. 64 The off-target effect can possibly cause harmful events such as sequence mutation, deletion, rearrangement, immune response, and oncogene activation, which limits the application of the CRISPR/Cas-9 editing system for therapeutic purposes. 65 To mitigate the possibility of CRISPR/Cas-9 off-target effect, several strategies have been developed, such as optimization of sgRNA, modification of Cas-9 nuclease, utilization of other Cas-variants, and the use of anti-CRISPR proteins. 66 Selecting and designing an appropriate sgRNA for the targeted DNA sequence is an important first step to reduce the off-target effect. 67 When designing sgRNA, strategies such as GC content, sgRNA length, and chemical modifications of sgRNA must be considered. Generally speaking, studies revealed that GC content of between 40% and 60%, truncated (short length of sgRNA), and incorporation of 2ʹ-O-methyl-3ʹ-phosphonoacetate in the sgRNA ribose-phosphate backbone are the preferred methods to increase genome editing efficiency of CRISPR/Cas-9. 67 , 68 Modifying the Cas-9 protein to optimize its nuclease specificity is another way to reduce off-target effects. For instance, mutating either one of the catalytic residues of Cas-9 nuclease (HNH and RuvC) will convert the Cas-9 into nickase that could only generate a single-stranded break instead of a blunt cleavage. 69 It has been reported that the use of the inactivated RuvC domain of Cas-9 with sgRNA can reduce the off-target effect by 100 to 1500 times. 70 Moreover, the nuclease Cas-12a (previously known as Cpf1) is a type V CRISPR/Cas system that provides high genome editing efficiency. 71 Unlike the CRISPR/Cas-9 system, CRISPR/Cas-12a can process pre-crRNA into mature crRNA without tracrRNA, thereby reducing the size of plasmid constructs. The Cas-12a protein recognizes a T-rich (5ʹ-TTTN) PAM sequence instead of 5ʹ-NGG and provides high accuracy at the target gene loci than Cas-9. 69 Recently, the use of multicomponent Class I CRISPR proteins, such as CRISPR/Cas-3 and CRISPR/Cas-10 provides better genome editing efficiency than Cas-9. 72 The Cas-3 is an ATP-dependent nuclease/helicase that can delete a large part of DNA from the target site without prominent off-target effect. For instance, the DMD gene were repaired by Cas-3-mediated system in induced pluripotent stem cell. 73 The Cas-10 protein does not require the PAM sequence and can identify sequences even in the presence of point mutation. 72 Anti-CRISPR (Acr) proteins are phage derived small proteins that inhibit the activity of CRISPR/Cas system. They are a recently discovered method to reduce off-target effects of CRISPR/Cas-9. 74 From Acr proteins, AcrIIA4 specifically targets Cas-9 nuclease. AcrIIA4 mimics DNA and binds to the Cas-9 site, making impossible to perform further cleavage in area outside the target region. 75 Furthermore, CRISPR/Cas-9 gene editing has been challenged by ethics and safety all over the world. Since the technology is still in its infancy and knowledge about the genome is limited, many scientists restrain that it still needs a lot of work to increase its accuracy and make sure that changes made in one part of the genome do not have unforeseen consequences, especially in the application towards human trials. 52

Conclusions

CRISPR/Cas-9 system in nature is used to protect prokaryotes from invading viruses by recognizing and degrading exogenous genetic elements. CRISPR/Cas-9 gene editing is adopted from acquired immunity in prokaryotes and consists of two elements: guide RNA used to locate (bind) the target DNA to be edited and Cas-9, a protein that essentially cuts the DNA at the location identified by guide RNA. The fundamental part of the CRISPR/Cas-9 gene-editing process is the identification of the target gene that determines the phenotype of interest and designing the guide RNA. Now it becomes a new era in molecular biology and has countless roles ranging from basic molecular researches to clinical applications. Although tremendous efforts have been made, there are still some challenges to rub in the practical applications and various improvements are needed to overcome obstacles in order to assure its maximum benefit while minimizing the risk.

Funding Statement

No funding was received.

Abbreviations

AAVs, adeno-associated viral vectors; ABE, adenine base editor; Acr, anti-CRSPR; AVs, adeno-viral vectors; ATP, adenosine tri-phosphate; BCL11A, B-cell lymphoma 11 A; CAS-9, CRISPR-associated protein-9; CBE, cytidine base editor; CCR5, chemokine receptor type 5; CFTR, cystic fibrosis conductance transmembrane receptor; CRISPR, clustered regularly interspaced short palindromic repeat; CrRNA, CRISPR ribonucleic acid; DMD, Duchenne muscular dystrophy; DNA, deoxyribonucleic acid; DSBs, double-stranded breaks; HDR, homology-directed repair; LVs, lentivirus vectors; NHEJ, non-homologous end Jjining; PAM, protospacer adjacent motif; PD-1, programmed cell death-1; RNA, ribonucleic acid; TALENs, transcriptionactivator like effector nucleases; TRAC, T-cell receptor alpha; TRBC, T-cell receptor beta; TracrRNA, trans-activating CRISPR ribonucleic acid; ZFNs, zinc finger nucleases.

Ethics Approval and Consent to Participate

Not applicable.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

The authors declare that they have no conflicts of interest for this work.

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You don’t win friends with bad salad! A gene editing approach to enhance the powdery mildew resistance in cucumber

Conflict of interest statement. None declared.

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Sara Selma, You don’t win friends with bad salad! A gene editing approach to enhance the powdery mildew resistance in cucumber, Plant Physiology , Volume 195, Issue 2, June 2024, Pages 908–910, https://doi.org/10.1093/plphys/kiae160

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One of the biggest challenges for agriculture lies in finding strategies that minimize crop yield loss due to pests and diseases. Powdery mildew (PM) is a widespread fungal disease that affects a diverse range of crops; for example, in cucumber ( Cucumis sativus L. ), PM can cause losses of up to 40% ( He et al. 2022 ). Various studies have focused on identifying PM resistance (PMR) genes that benefit cucumber breeding programs ( Liu et al. 2008 ). The characterization of quantitative trait loci (QTL) for mapping PMR linked the disruption of the Mildew resistance Locus 8 ( CsMLO8 ) gene with the PM resistance in cucumber. However, although the loss of function of CsMLO8 is indispensable for the PMR, it is not enough to generate a complete resistance ( Nie et al. 2015a , 2015b ; Berg et al. 2015 ). The PM-resistant QTL also contains other members of the CsMLO family, pointing out that more than 1 CsMLO gene is involved in PM resistance ( Schouten et al. 2014 ). A recent study characterized the MLO proteins as calmodulin-gated calcium channel proteins ( Gao et al. 2022 ), suggesting that calcium signaling is involved in mlo -mediated PM resistance. However, both the components and mechanism of PM resistance remain not fully understood.

In this issue of Plant Physiology , Ma et al. 2024 identify the CsMLO genes involved in the resistance against PM infection. In addition, they validated the link between the Csmlo- mediated PM resistance and the calcium signaling pathway through the identification of novel calcium-related proteins in the PM resistance.

As a starting point, the authors evaluated the potential role of the CsMLO family in PM resistance by mutating all the CsMLO members using CRISPR/Cas9-based multiplex gene editing ( Čermák et al. 2017 ). The CRISPR-Cas9 system is a widespread method for genome editing that consists of a Cas9 protein, which acts as a pair of “molecular scissors” of the target DNA and a small piece of RNA (gRNA) that drives the Cas9 to the target DNA sequence. The CsMLO family contains 13 CsMLO genes divided into 5 different clades ( Fig. 1A ), so the authors optimized the CRISPR-Cas9 system to perform an efficient multiplexing approach in cucumber that allows the expression of several gRNAs at the same time. Eight-target, 3-target, and 2-target constructs were designed to mutate the 13 CsMLO members ( Fig. 1A ), obtaining 17 independent T0 transgenic plants with high editing efficiency. Csmlo mutants were generated in less than 2 generations, highlighting the success of the multiplexed gene editing system developed by the authors.

Identification of the CsMLO genes involved in the resistance against PM infection. A) Clade classification of the CsMLO genes accompanied the phenotypic observation of the Csmlo -mutated plants compared to WT. The green-colored box represents the genes edited with 2-target construct. The red-colored box represents the genes edited with 3-target construct. The blue-colored box represents the genes edited with 8-target construct. No loss-of-function mutation was obtained from the CsMLO2 , C sMLO7 , and CsMLO6 genes. B) PM resistance phenotypes of WT and clade V Csmlo mutants. The grayish-white powder on the leaves indicates the presence of the fungus. The phenotypes were documented at 20 days post infection (dpi) in the greenhouse. Figure extractect from Ma et al. 2024 .

The growth phenotypes of Csmlo mutants were assessed to point out the function of the CsMLO clades but also potential pleiotropic effects that might alter the yield and quality of the crops. The results show that the 8-target construct employed to assess clades I, II, III, and VI resulted in the frameshift mutations of Csmlo5 , Csmlo3 , Csmlo3/10 , Csmlo3/5/10 , Csmlo3/4/5/10 , and Csmlo3/4/5/9/10 . Notably, flowers from these mutants showed an increased proportion of petals and higher carpel numbers compared to the wild-type (WT). On the other hand, the Csmlo12/13 mutant exhibited larger leaves than the WT and male sterility due to hindered pollen germination. Finally, the Csmlo1/8/11 triple mutants (clade V) presented smaller leaves, reduced plant height, and shorter fruits compared to the WT. The results highlight the significant role of the CsMLO family in diverse developmental stages.

Regarding PM resistance in the Csmlo plants, the results show that the Csmlo8 single mutant was fully susceptible to PM, with no significant difference compared to the WT. Interestingly, the double mutants Csmlo1/8 and Csmlo8/11 showed moderate resistance, and the triple Csmlo1/8/11 exhibited complete PM resistance ( Fig. 1B ). These results support the previous studies that concluded that CsMLO8 disruption was insufficient but necessary to generate PM resistance, demostrating a functional redundancy among clade V. The rest of the CsMLO loss-of-function lines were equally susceptible than the WT to PM, indicating that this function is restricted to the CsMLO clade V in cucumber.

Additionally, the authors performed a transcriptomic and proteomic analysis to unravel the molecular mechanisms underlying PM resistance using the susceptible WT, Csmlo1/8 with moderate PM resistance, and Csmlo1/8/11 with complete PM resistance. The differential expression analysis comparing the PM-susceptible and PM-resistant genotypes matches with the proteomics results revealing a functional enrichment of genes involved in “plant-pathogen interaction” and “calcium ion binding.” The candidates KCBP-interacting Ca 2+ binding protein (CsKIC), CaM-like protein 28 (CsCML28), and Ca 2+ − dependent protein kinase 11 (CsCPK1) were selected from the enriched “calcium ion binding” category as potential new players involved in the PM resistance ( Kim et al. 2002 ; Freymark et al. 2007 ). CsKIC exhibited both mRNA and protein upregulation in Csmlo1/8/11 , while CsCML28 and CsCPK11 showed drastic protein changes without significant mRNA alterations, suggesting a possible post-transcriptional and post-translational regulation. Yeast-2-hybrid assays reveal that CsKIC can interact with the C terminus of CsMLO8 but also with the C termini of CsMLO1 and CsMLO11, indicating possible collaborative functions. No interactions were observed between CsKIC, CsCML28, and CsCPK11.

To further investigate the role of CsKIC , CsCML28 , and CsCPK11 in cucumber’s resistance to PM, a virus-induced gene silencing approach was employed to downregulate their expression. Silencing CsKIC and co-silencing of CsKIC with CsMLO8 resulted in enhanced PM resistance, indicating that CsKIC acts with CsMLO8 as a negative regulator of PM resistance. On the contrary, silencing CsCML28 and CsCPK11 increased susceptibility to PM, indicating that they act as positive regulators in PM resistance.

In summary, the work by Ma et al. 2024 uncovered genes involved in cucumber PM resistance. Although the triple mutant Csmlo1/8/11 is completely resistant to the PM infection, it shows significant growth penalties not desired in the breeding programs. In addition, the role of the Ca 2+ binding proteins CsKIC, CsCML28 , and CsCPK11 in the Csmlo -mediated PM resistance could offer new strategies to obtain PM-resistant varieties. However, this calcium-dependent mechanism is still not completely understood. Future studies may focus on that direction to benefit not only cucumber breeding programs but other PM-sensitive crops.

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