Genetic editing goes rogue
Story at-a-glance -
- Emerging research suggests gene editing may not be as precise as once thought and may lead to significant off-target mutations, with completely unknown consequences
- Researchers from Massachusetts General Hospital (MGH) have revealed that CRISPR base editors, which are intended to target a single DNA base, induce widespread off-target effects in RNA
- Tens of thousands of base changes were revealed as a result of CRISPR-Cas base editor technology; the widespread RNA changes led to mutations in protein-coding and noncoding sequences
- The benefits of gene editing, such as potentially providing a cure for various blood disorders, lung diseases and cancers, are so alluring that scientists are moving full-speed ahead without stopping to fully assess whether or not they should
Advancements in genetic editing claim to offer a precise way to modify an organism's DNA, opening new doors to treat diseases, improve food crops, manufacture fuel and chemicals and more. The possibilities seem virtually endless, but emerging research suggests the edits may not be as precise as once thought and may lead to significant off-target mutations, with completely unknown consequences.
As increasing off-target implications are uncovered from gene-editing tools such as CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeat, and TALEN (Transcription Activator-Like Effector Nuclease), the reality is the opposite of what was advertised: the perfectly precise snips to DNA may not be so exact after all, highlighting the complete lack of understanding of the complexities that come with attempting to alter genetics.
CRISPR Gene Editing Leads to Widespread Unintended Changes to RNA
Researchers from Massachusetts General Hospital (MGH) have revealed that CRISPR base editors, which are intended to target a single DNA base, induce widespread off-target effects in RNA.1 Dr. J. Keith Joung, the study's senior author, explained in a news release:2
"Most investigation of off-target base editing has focused on DNA, but we have found that this technology can induce large numbers of RNA alterations as well … This surprising finding suggests the need to look at more than just genetic alterations when considering unintended off-target effects of base editors in cells.
We also show the feasibility of reducing these effects by creating variants that selectively reduce off-target RNA editing while preserving the intended on-target DNA activity."
Tens of thousands of base changes were revealed as a result of CRISPR-Cas base editor technology. The widespread RNA changes led to mutations in protein-coding and noncoding sequences.3 "We were quite surprised at the number — tens of thousands — of RNA edits and the frequency of these alterations that we observed with the two classes of base editors," study author Dr. Julian Grünewald, MGH Molecular Pathology and Harvard Medical School stated.4
As for the consequences of these unintended changes, the team plans to study any potential impact next, but suggests such repercussions should be taken into account in safety assessments of the technology.
How Do CRISPR and CRISPR Cas-9 Work?
Whereas gene editing was once a very imprecise and expensive process, scientists can now go into your DNA and essentially cut and paste it at specified places. The technology can be traced back to bacteria, which protect themselves by cutting out invading virus' DNA and inserting it into their own, then replicating the new sequences to prevent future viral invasions.5
In 2012, researchers refined the system and revealed that any DNA (not just bacteria) has this ability — and the process works in humans.6 With CRISPR-Cas 9, the technology was said to be even more precise, acting as a pair of scissors to "snip" DNA at specific locations. As explained by the U.S. National Institutes of Health:7
"CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. The bacteria capture snippets of DNA from invading viruses and use them to create DNA segments known as CRISPR arrays. The CRISPR arrays allow the bacteria to 'remember' the viruses (or closely related ones).
If the viruses attack again, the bacteria produce RNA segments from the CRISPR arrays to target the viruses' DNA. The bacteria then use Cas9 or a similar enzyme to cut the DNA apart, which disables the virus.
… Although Cas9 is the enzyme that is used most often, other enzymes (for example Cpf1) can also be used. Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence."
In the featured study, researchers looked into CRISPR base editors, which they compared to a pencil, as opposed to scissors. CRISPR base editors use deaminase, an enzyme, to modify specific nucleotides, which are the basic structural units of DNA.
Unexpected Mutations Common With Gene Editing
Off-target mutations seem to be the rule rather than the exception when it comes to genetic editing. This flaw isn't unheard of, and it's been tested for before, typically using a computer algorithm to predict where such off-target mutations are likely to occur, then searching those areas to see if such mutations did, in fact occur.
However, one study used a different method to search for unintended mutations, based on a separate study that used CRISPR-Cas9 to restore sight in blind mice by correcting a genetic mutation.
The researchers sequenced the entire genome of the CRISPR-edited mice to search for mutations. In addition to the intended genetic edit, they found more than 100 additional deletions and insertions along with more than 1,500 single-nucleotide mutations, raising concerns that testing CRISPR in humans may be premature, even with CRISPR-Cas 9.8
Study author Dr. Stephen Tsang of Columbia University Medical Center said that even a single change to a nucleotide could have a "huge impact."9 Indeed, in animals, gene editing has led to unexpected side effects, including enlarged tongues and extra vertebrae.10,11
The fact is, researchers typically don't know the extent of a gene's functions until they attempt to tweak it, and something like an extra vertebra reveals itself. Speaking with Yale Insights, Dr. Greg Licholai, a biotech entrepreneur, explained some of the very real risks of CRISPR and other gene-editing technologies:12
"One of the biggest risks of CRISPR is what's called gene drive, or genetic drive. What that means is that because you're actually manipulating genes and those genes get incorporated into the genome, into the encyclopedia, basically, that sits within cells, potentially those genes can then be transferred on to other organisms.
And once they're transferred on to other organisms, once they become part of the cycle, then those genes are in the environment. That's probably the biggest fear of CRISPR. Humans manipulating the genetic code, and those manipulations get passed on generation to generation to generation.
We think we know what we're doing, we think we're measuring exactly what changes we're doing to the genes, but there's always the possibility that either we miss something or our technology can't pick up on other changes that have been made that haven't been directed by us."
Do the Risks of Gene Editing Outweigh the Benefits?
The benefits of gene editing, such as potentially providing a cure for various blood disorders, lung diseases and cancers, are so alluring that scientists are moving full-speed ahead without stopping to fully assess whether or not they should. Particularly in the case of editing germline cells, such as embryos, eggs and sperm, changes made to the genome will be inherited by future generations.
While experts have previously said CRISPR and Cas 9 should not be used on human babies, a report released in February 2017 by the National Academies of Sciences and Medicine stated DNA in germline cells may be altered to eliminate genetic diseases.13
The stipulation was that the technology be used only to correct disease or disability, not enhance health or ability.14 Many support the use of gene-editing technology for the purpose of eliminating genetic diseases, but what about nondisease conditions like poor impulse control to increase a child's opportunities in life?
"There is, for example, the risk that the introduction and eventual wide utilization of gene editing technology will exacerbate existing inequalities resulting in human rights abuses, a new wave of eugenics, increased discrimination and increased stigmatization," writes bioethicist Francoise Baylis of Dalhousie University in Canada, in Clinical Chemistry.15
She believes that the primary risks of CRISPIR Cas 9 are based on the broadening of social and economic inequalities that could result if "designer babies" and the use of gene editing for nonmedical purposes become commonplace. Bavlis explains:16
"As such, the overarching risks with human gene editing by use of CRISPR-Cas9 are two-fold. First, there is the risk that certain social, economic, and political forces will come to bear on those deemed "unfit" in an effort to pressure them to change their genetics so that they might better conform to certain external norms or expectations. Second, there is the risk that those who resist pressure to conform will experience (further) oppression."
Gene Editing May Trigger Tumor Growth
Off-target mutations that occur as the result of gene editing may take on many forms, including rearranging chromosomes, inactivating essential genes or improperly activating others, such as cancer-causing genes.17
For instance, CRISPR-Cas 9 leads to the activation of the p53 gene, which works to either repair the DNA break or kill off the CRISPR-edited cell.18 CRISPR actually has a low efficacy rate for this reason, and CRISPR-edited cells that survive are able to do so because of a dysfunctional p53.
The problem is that p53 dysfunction is also linked to cancer (including close to half of ovarian and colorectal cancers and a sizable portion of lung, pancreatic, stomach, breast and liver cancers as well).19
In one study, researchers were able to boost average insertion or deletion efficiency to greater than 80 percent, but that was because of a dysfunctional p53 gene,20 which would mean the cells could be predisposed to cancer.
The researchers noted, " … it will be critical to ensure that [CRISPR-edited cells] have a functional p53 before and after engineering."21 A second study, this one by the Karolinska Institute in Sweden, found similar results and concluded, " … p53 function should be monitored when developing cell-based therapies utilizing CRISPR–Cas9."22
Some have suggested that if CRISPR could cure one chronic or terminal disease at the "cost" of an increased cancer risk later,23 it could still be a beneficial technology, but most agree that more work is needed and caution warranted.
Gene Editing Is Uncharted Territory
Gene editing is here to stay and is quickly moving from the medical realm to agricultural. A gene-edited soybean oil created by biotech company Calyxt hit the market in 2019 and is being used by a Midwest company with both restaurant and foodservice locations for frying as well as in dressings and sauces.
In food, off-target edits could cause unintended changes to plant DNA, with consequences that could include growth disturbances, exposure to plant diseases or the introduction of allergens or toxins.24 Further, although they're genetically engineered, gene-edited foods are not marketed as GMOs, nor are they labeled as such.
So choosing organic or biodynamic is the only way to ensure that gene-edited oils aren't making it onto your dinner table. Low-gluten wheat, mushrooms that don't turn brown and tomatoes that can be produced in areas with shorter growing seasons are among the products created using genetic editing.
While the European Union has ruled that gene edited crops must go through the same approval process as GMOs, Japan recently concluded that such foods could enter the consumer marketplace without any safety studies, as did the U.S.
While the potential benefits are driving gene editing research forward at an unprecedented pace, studies show that tinkering with genetics is far more complex than making a snip here and an insertion there.
Adding in the potential ethical considerations, an article in Religions recommends scientists invoke the Precautionary Principle, using due caution but proceeding with proper risk assessments. However, it also posits:25
"Human civilization must critically examine the scientific (technological) imperative. Simply because society can pursue a particular medical, reproductive or genetic procedure does not mandate that it must!
Especially in the area of genetics, 'can' does not mandate 'ought.' The potential for power and control and its obvious abuse mandates an examination of this imperative. Perhaps with some of these procedures, such as gene editing, it would be wise to not do them at all."
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