Computer illustration showing the molecular structure of the CRISPR-associated protein Cas9 in complex with RNA (ribonucleic acid, red) and DNA (deoxyribonucleic acid, orange-fuchsia). Cas9 is a nuclease protein associated with the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes and other bacteria. It uses a guide RNA sequence to cut DNA at a complementary site. Seen here: Cas9 alpha-helical lobe (grey), HNH domain (blue), RuvC domain (light blue), C-terminal-domain (pink), topo homology domain (pale pink), arginine-rich helix (light fuchsia), guideRNA (red), target DNA (orange), non-target DNA (fuchsia). | Ramon Andrade 3DCiencia/Science Photo Library/Getty Images

CRISPR DNA Editing Can Cause Hundreds of Off-Target Mutations

A new study offers a cautionary tale for using the widely hyped gene-editing tool CRISPR on people.

Published On 06/06/2017
9:32 PM EDT
T T he gene-editing technology known as CRISPR has only been around for five years, but it’s already generated enough hype and controversy to last a lifetime. Compared to other gene-editing techniques, CRISPR is so fast, cheap, and precise that any scientist with a basic understanding of genetics can experiment with DNA — plant, animal, or human — by cutting out and replacing specific genes.
Some say that the ease and effectiveness of CRISPR will usher in a golden age of genetic modification resulting in new cures for inherited diseases like Huntington’s and muscular dystrophy and new gene therapies for eradicating cancer and HIV. Others see CRISPR as a Pandora’s box leading to a dystopian future of designer babies and expensive life-extending therapies that will further divide the world into genetic haves and have nots.
Now a new study complicates the CRISPR controversy by shining a light on CRISPR’s Achilles' heel — off-target mutations. CRISPR works by targeting short sequences of DNA base pairs in the genome (combinations of the nucleotides A, C, G, T), but researchers have long worried that CRISPR’s molecular “scissors” could accidentally snip other sites on the genome that contain the same or similar sequence. Until now, most scientists believed they could accurately predict the likelihood of off-target mutations using advanced algorithms that look for repeats in the code.
But last week, researchers from the Columbia University Medical Center, the University of Iowa, and Stanford University published a paper in the journal Nature Methods citing more than 1,500 small off-target mutations and more than 100 “larger insertions and deletions” in the DNA of mice that had been treated with CRISPR. Instead of relying on predictive algorithms, the scientists did side-by-side comparisons of fully sequenced genomes from the CRISPR-treated mice and genomes from “genetically identical” mice from the same colony.
“We started seeing mutations in lots of different places that weren’t predicted at all by the algorithms,” said Vinit Mahajan, co-author of the study and a professor of ophthalmology at Stanford. “When you read about CRISPR, the focus is on the amazing ability of this system to go in and fix a single nucleotide out of the three billion in a genome. People have been looking at off-targeting, but generally you get the sense that it’s not that bad, or it’s not consequential. So this definitely came as a surprise.”
The revelation that CRISPR, short for Clustered Regularly Interspersed Palindromic Repeats, can accidentally edit both single nucleotides and larger chunks of DNA in living animals comes at a pivotal moment for the controversial technology. Because of its low cost and high speed, CRISPR has quickly become the go-to gene-editing tool for basic scientific research. More recently, it’s made the leap from the lab to the hospital as physicians look for ways to boost the efficacy of gene therapies for diseases like cancer.
In that type of gene therapy, scientists attempt to edit the DNA of a patient’s own immune cells (T cells) so they can better target and hunt down the specific type of cancer attacking the body. Physicians have been experimenting with different gene-editing methods including zinc finger nucleases and TALENs, but those technologies are thought to be less precise — and a lot slower and more expensive — than CRISPR.
In 2016, the very first clinical trial was approved in the United States to use CRISPR on human T cells that will be transplanted back into a cancer patient. Dean Anthony Lee is a pediatric oncologist and cell therapist who sits on the NIH committee that gave the green light to the first CRISPR trial on humans. Lee said that the recent off-targeting study confirms what the research community has long suspected, that CRISPR is the “most leaky” of the available gene-editing technologies when it comes to accidental edits.
“But it didn’t present anything new that all of us went, ‘Oh no, what did we do?’” Lee said.
Instead, said Lee, the off-target study made it clear that CRISPR still carries some unknown risks, and that it should only be applied clinically in select situations where the individual “really doesn’t have any other options,” like with a relapsed cancer that will probably take a patient’s life within months or even weeks.   
“In a setting like that, where you’ve already got a disease that’s likely to kill you, that might be a reasonable risk to take, as long as it’s spelled out to the patient what the potential outcomes could be,” said Lee. He sees CRISPR-based gene therapy as the latest in a long line of last-hope cancer treatments that carried great risks in the outset. The first chemotherapy drugs, after all, were derived from mustard gas.   
Mahajan, co-author of the off-targeting study, is also an eye surgeon and said that he’s still “very enthusiastic about CRISPR.” The reason he and his team had access to the mouse genomes was because they had recently used CRISPR to restore sight to three blind mice (no relation to the nursery rhyme). His small study wasn’t meant to be the “nail in the coffin” for CRISPR, said Mahajan, but rather a “cautionary tale.” He hopes that the research community will explore off-target edits further so that physicians like himself can explain the true risks of CRISPR-based therapies to their patients.
Marcy Darnovksy from the Center for Genetics and Society — a nonprofit that advocates for the ethical and equitable use of genetic and reproductive technologies — agreed that the off-targeting study struck a “needed note of caution.” She worries that because CRISPR is so much easier to use, and so much faster and cheaper, that it “creates a temptation for everyone to pick it up and use it for everything.”
Darnovsky is less concerned with the clinical use of CRISPR to treat advanced cancers and HIV than with the potential for using CRISPR to precision-edit human embryos. There are more than 40 countries worldwide that make it a criminal offense to modify inheritable traits in human embryos. The United States, interestingly, is not one of them. Although current regulations forbid using federal dollars to fund such controversial research, the door isn’t officially closed.
In fact, Darnovsky pointed out, the National Academies of Science and Medicine issued a report in February that explicitly left the door open for so-called “germline editing” in cases of serious genetic diseases for which “no reasonable alternative” existed. The report called for stringent oversight of such research and a strict set of criteria to separate true diseases from genetic “enhancements,” but for Darnovsky, the potential risks clearly outweigh the benefits.
“I don’t know if there’s ever been a risk quite like this,” she said, referring to the possibility of a society dominated by genetic superiors.This isn’t a door that you can open just a crack. Once you open it at all, it gets blown wide open.”
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