CRISPR Gene Editing Caused Hundreds of Unintended Mutations in Mice Genome

THIRD WORLD NETWORK BIOSAFETY INFORMATION SERVICE

Dear Friends and Colleagues

CRISPR Gene Editing Caused Hundreds of Unintended Mutations in Mice Genome

As CRISPR-Cas9, a new gene-editing technology, moves into gene therapy clinical trials, a new study published in Nature Methods has reported that it can introduce hundreds of unintended mutations into the genome of mice, including single nucleotide mutations and mutations in non-coding regions of the genome (Item 1). The first clinical trial to deploy CRISPR is now underway in China, and a U.S. trial is slated to start next year.

Even though CRISPR targets specific stretches of DNA, it sometimes hits other parts of the genome. Whole genome sequencing has, however, not been employed to look for all off-target effects in living animals. In the new study, the researchers sequenced the entire genome of mice that had undergone CRISPR gene editing in the team’s previous study and looked for all mutations, including those that only altered a single nucleotide. The researchers found that the genomes of two independent gene therapy recipients had sustained more than 1,500 single-nucleotide mutations and more than 100 larger deletions and insertions. None of these DNA mutations had been predicted by computer algorithms that are widely used by researchers to look for off-target effects.

This new study shows that CRISPR is not as precise or predictable as claimed. So how should food plants and animals derived from CRISPR and other genome editing techniques be assessed? Dr Michael Antoniou, a London-based molecular geneticist, believes that not only is it necessary to conduct whole genome sequencing to identify all off-target mutations from CRISPR-based genome editing, but it is also essential to ascertain the effects of these unintended changes on global patterns of gene function (Item 2).

Therefore one needs to follow up whole genome sequencing with other molecular profiling analyses or “omics”: transcriptomics – gene expression profiling, proteomics – protein composition profiling, metabolomics – profiling of metabolites, and miR-omics – microRNA profiling. In addition, it is important to acknowledge that the targeted intended change in a given gene may also have unintended effects. For example, the total disruption or modification of an enzyme function can lead to unexpected or unpredictable biochemical side-reactions that can markedly alter the composition of an organism, such as a food crop. Finally, Dr Antoniou stresses the necessity of conducting long-term toxicity studies in established animal model systems.

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Item 1

CRISPR GENE EDITING CAN CAUSE HUNDREDS OF UNINTENDED MUTATIONS

Columbia University Medical Center
May 30, 2017
http://newsroom.cumc.columbia.edu/blog/2017/05/30/crispr-gene-editing-can-cause-hundreds-of-unintended-mutations/

New York—As CRISPR-Cas9 starts to move into clinical trials, a new study published in Nature Methods has found that the gene-editing technology can introduce hundreds of unintended mutations into the genome.

“We feel it’s critical that the scientific community consider the potential hazards of all off-target mutations caused by CRISPR, including single nucleotide mutations and mutations in non-coding regions of the genome,” says co-author Stephen Tsang, MD, PhD, the Laszlo T. Bito Associate Professor of Ophthalmology and associate professor of pathology & cell biology in the Institute of Genomic Medicine and the Institute of Human Nutrition at Columbia University Medical Center.

CRISPR-Cas9 editing technology—by virtue of its speed and unprecedented precision—has been a boon for scientists trying to understand the role of genes in disease. The technique also has raised hope for more powerful gene therapies that can delete or repair flawed genes, not just add new genes.

The first clinical trial to deploy CRISPR is now underway in China, and a U.S. trial is slated to start next year. But even though CRISPR can precisely target specific stretches of DNA, it sometimes hits other parts of the genome. Most studies that search for these off-target mutations use computer algorithms to identify areas most likely to be affected and then examine those areas for deletions and insertions.

“These predictive algorithms seem to do a good job when CRISPR is performed in cells or tissues in a dish, but whole genome sequencing has not been employed to look for all off-target effects in living animals,” says co-author Alexander Bassuk, MD, PhD, professor of pediatrics at the University of Iowa.

In the new study, the researchers sequenced the entire genome of mice that had undergone CRISPR gene editing in the team’s previous study and looked for all mutations, including those that only altered a single nucleotide.

The researchers determined that CRISPR had successfully corrected a gene that causes blindness, but Kellie Schaefer, a PhD student in the lab of Vinit Mahajan, MD, PhD, associate professor of ophthalmology at Stanford University, and co-author of the study, found that the genomes of two independent gene therapy recipients had sustained more than 1,500 single-nucleotide mutations and more than 100 larger deletions and insertions. None of these DNA mutations were predicted by computer algorithms that are widely used by researchers to look for off-target effects.

“Researchers who aren’t using whole genome sequencing to find off-target effects may be missing potentially important mutations,” Dr. Tsang says. “Even a single nucleotide change can have a huge impact.”

Dr. Bassuk says the researchers didn’t notice anything obviously wrong with their animals. “We’re still upbeat about CRISPR,” says Dr. Mahajan. “We’re physicians, and we know that every new therapy has some potential side effects—but we need to be aware of what they are.”

Researchers are currently working to improve the components of the CRISPR system—its gene-cutting enzyme and the RNA that guides the enzyme to the right gene—to increase the efficiency of editing.

“We hope our findings will encourage others to use whole-genome sequencing as a method to determine all the off-target effects of their CRISPR techniques and study different versions for the safest, most accurate editing,” Dr. Tsang says.

Item 2

CRISPR-INDUCED MUTATIONS – WHAT DO THEY MEAN FOR FOOD SAFETY?

Claire Robinson
1 June 2017
http://www.gmwatch.org/en/news/latest-news/17657

A new study published in Nature Methods has found that the genome editing technology CRISPR introduced hundreds of unintended mutations into the genome of mice.[1]

In the study, the researchers sequenced the entire genome of mice that had undergone CRISPR gene editing to correct a genetic defect. They looked for all mutations, including those that only altered a single nucleotide (DNA base unit).

They found that the genomes of two independent gene therapy recipients had sustained more than 1,500 single-nucleotide mutations and more than 100 larger deletions and insertions. None of these DNA mutations were predicted by the computer algorithms (software packages) that are widely used by researchers to screen the genome (the total DNA base unit sequence) of an organism to look for potential off-target effects.

While this study was conducted in the arena of gene therapy, it has clear implications for the regulation of food plants and animals derived from CRISPR and other genome editing techniques.

Regulatory agencies across the world are currently engaged in a debate about how to assess genome-edited products for safety. Many GMO proponents are proposing “light-touch” regulation or even no regulation at all, based on the assumption that the outcome genome editing techniques like CRISPR are precise, predictable, and therefore safe.

The new study shows that this assumption is false. So how should these products be regulated?

One suggestion that has been put forward is to require whole genome sequencing of gene-edited organisms to be conducted and submitted to biosafety authorities.

But this raises a further question: if the whole genome sequence does not show any mutations or off-target effects, other than those intended, should we be reassured?

We asked Dr Michael Antoniou to comment. Dr Antoniou is a London-based molecular geneticist who uses genetic engineering techniques, including genome editing, to develop gene therapies.

Dr Antoniou says:

I agree that the whole genome sequences of gene-edited organisms must be submitted to biosafety authorities. And if the whole genome sequence did not show any additional mutations/off-target effects other than those intended, this would be somewhat reassuring.

However, it is highly unlikely that this will ever be the case. It is a matter of how many off-target mutations there are, rather than a matter of their total absence. The technology is not perfect. It will in future become less prone to off-target effects as it is refined procedurally, but it is extremely unlikely that it will ever arrive at a point where only the intended change will result.
 
In addition, the application of genome editing technologies in an agricultural context will involve plant tissue culture, which has its own inherent mutagenic properties. So no matter how precise the genome editing becomes, there will always be a large spectrum of tissue culture-induced mutations present.
 
Many of the genome editing-induced off-target mutations, as well as those induced by the tissue culture, will no doubt be benign in terms of effects on gene function. However, many will not be benign and their effects can carry through to the final marketed product, whether it be plant or animal.

There is an additional feature that makes genome editing more likely to bring about off-target gene damage resulting in a disturbed function. This stems from the fact that the off-target effects will not be random, but will take place at sites within other genes that are similar in DNA base unit sequence to where the intended change has taken place.

This does not exclude the recent and unsurprising finding from the recent study in mice, which found that off-target effects from CRISPR can occur at sites within the genome whose DNA base unit sequence is markedly dissimilar from the targeted site. The researchers found that the large numbers of off-target mutations caused by CRISPR in mice could not be predicted by the usual computer algorithms.[1]
 
Thus not only is it necessary to conduct whole genome sequencing to identify all off-target mutations from CRISPR-based genome editing, but it is also essential to ascertain the effects of these unintended changes on global patterns of gene function. Therefore one needs to follow up the whole genome sequencing with other molecular profiling analyses or “omics”: transcriptomics — gene expression profiling, proteomics — protein composition profiling, metabolomics — profiling of metabolites, and miR-omics – microRNA profiling.
 
In addition, it is important to acknowledge that the targeted intended change in a given gene may also have unintended effects. For example, the total disruption or modification of an enzyme function can lead to unexpected or unpredictable biochemical side-reactions that can markedly alter the composition of an organism, such as a food crop.
 
The compositional alterations in food products produced with genome editing techniques will not be fully revealed by the molecular profiling methods due to the current inherent limitations of these techniques. So it is still necessary to conduct long-term toxicity studies in established animal model systems.
 
In the absence of these analyses, to claim that genome editing is precise and predictable is based on faith rather than science.

GMWatch’s conclusion

We conclude from Dr Antoniou’s explanation that the products of genome editing techniques should be at least as stringently regulated as the products of old-style GM techniques. In fact, regulation for all types of GM products should be overhauled to include “omics” molecular analyses and long-term animal feeding studies. Such analyses and studies are not currently demanded by any regulatory authority anywhere in the world.

References
1. Schaefer KA, Wu W-H, Colgan DF, Tsang SH, Bassuk AG, Mahajan VB. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nat Methods. 2017;14(6)2017-06-13 13:50:2947-548. doi:10.1038/nmeth.4293.