It's not hyperbolic to say that the CRISPR-Cas9 gene-editing technique has been a revolutionary breakthrough, allowing scientists the ability to quickly, easily and precisely edit sections of DNA. But questions over how precise the CRISPR tool is have been raised in a new study from Columbia University Medical Center, which shows this gene-editing technology can introduce hundreds of unintended mutations into the genome.
CRISPR has sparked a flurry of new avenues of research around the world, from targeting cancer to HIV, with the first human trials involving CRISPR-edited cells already underway in China and a US trial slated for 2018. But this new study urges caution moving forward, suggesting we are still yet to understand the greater genomic effects of the tool.
The team of scientists involved in the study had previously been working with the CRISPR tool to treat a serious eye disease called retinitis pigmentosa, which leads to blindness. They decided to examine the entire genome of the CRISPR-treated mice from their previous experiments, looking for any potential mutations, even those that altered just a single nucleotide.
Generally, when scientists are trying to identify whether a CRISPR edit has resulted in an off-target mutation or deletion they use computer algorithms to identify areas most likely to be affected and focus their attention on those.
"These predictive algorithms seem to do a good job when CRISPR is performed in cells or tissues in a dish," says co-author of the study, Professor Alexander Bassuk, "but whole genome sequencing has not been employed to look for all off-target effects in living animals."
In examining the entire genome from the CRISPR-treated mice, they found that the tool had successfully corrected the specific gene they were targeting, but it also potentially caused a great deal of other genetic changes. In two CRISPR-treated animals, more than 100 large gene deletions or insertions and over 1,500 single-nucleotide mutations were identified.
Most significantly, all of these identified mutations were not picked up by the general computer algorithms most researchers use to look at the off-target effects of CRISPR-editing. There were no obvious or immediately deleterious effects in the animals from these unexpected mutations, but it is unknown what longer term effects the altered genes could have.
"Researchers who aren't using whole genome sequencing to find off-target effects may be missing potentially important mutations," says co-author Dr. Stephen Tsang. "Even a single nucleotide change can have a huge impact."
The team is still upbeat about CRISPR technology, but they caution other scientists to more closely study the off-target effects of any gene-editing that is undertaken. They especially note that whole-genome sequencing is vital in developing more accurate ways of using the CRISPR tool.
"We're physicians," explains co-author of the study Dr Vinit Mahajan, "and we know that every new therapy has some potential side effects but we need to be aware of what they are."
The study was published in the journal Nature Methods.