Patients’ individual genomes may affect efficacy, safety of gene editing

gene editing - truck delivering code
Subtle genetic variants in or near the gene editing target site could cause reagents to miss an address or arrive at the wrong one, researchers say.

Gene editing has begun to be tested in clinical trials, using CRISPR-Cas9, zinc finger nucleases (ZFN) and other technologies to directly edit DNA inside people’s cells. Multiple trials are in the recruiting or planning stages. But a study in PNAS this week raises a note of caution, finding that person-to-person genetic differences may undercut the efficacy of the gene editing process or, in more rare cases, cause a potentially dangerous “off target” effect.

The study adds to evidence that gene editing may need to be adapted to each patient’s genome, to ensure there aren’t variants in DNA sequence in or near the target gene that would throw off the technology.

“Humans vary in their DNA sequences, and what is taken as the ‘normal’ DNA sequence for reference cannot account for all these differences,” notes Stuart Orkin, MD, of Dana-Farber Boston Children’s Cancer and Blood Disorders Center and co-corresponding author on the study with Matthew Canver, an MD-PhD student at Harvard Medical School. “We recommend that common variation be taken into account in designing targeting systems for therapeutic editing, to maximize efficacy and minimize potential safety concerns.”

Search errors?

The research team, led by Orkin, Canver and Samuel Lessard of the University of Montreal, analyzed 7,444 previously published whole-genome sequences. They gathered a list of about 30 disease-related DNA targets that researchers are interested in altering through gene editing.

Next, they made a second list of nearly 3,000 guide RNAs (gRNAs). Like an address on an envelope, these bits of genetic code are designed to direct CRISPR-Cas9 enzymes to the right editing location.

Finally, the team looked to see whether any of the 7,444 individuals carried DNA sequence variants (“letter changes” or insertions/deletions) in the areas the gRNAs are looking for.

“If there are genetic differences at the site that CRISPR reagents are targeting for therapy, you are at risk for decreased efficacy or treatment failure,” explains Canver, who conceived and led the study in Orkin’s Boston Children’s Hospital lab. “A difference in just a single base pair can cause a decrease in binding efficiency due to a mismatch with the guide RNA. Overall, this can cause a reduction in treatment efficacy.”

One reagent doesn’t fit all

Such occurrences in the genome turned out not to be uncommon. In fact, about 50 percent of the analyzed gRNAs had the potential to be affected by variants at their target sites. In a few cases, the researchers found genetic variants that could potentially draw a gRNA to the wrong place — resulting in an edit of a gene or other DNA region that’s not meant to be targeted.

Genetic variation among CRISPR-Cas9 target regions may affect efficacy, safety of gene editing
Genetic variation among CRISPR-Cas9 target regions: At the top, a 30-base-pair region of a single genetic locus (HLA-A) that is involved in the immune response and could potentially be targeted with gene editing. Below it, the six smaller barsit represent different guide RNAs that are designed to bind to different parts of that HLA-A locus. On the Y (vertical) axis are haplotypes with highlighted DNA variations identified from different individuals. They are aligned according to their positions in the genetic sequence (represented horizontally on the X axis). (Lessard S; et al. PNAS Early Edition, week of Dec. 11, 2017)

“In rare cases, there was the potential to create very potent ‘off-target’ sites – where CRISPR reagents could bind and cut where they’re not intended to,” says Canver. “If an off-target effect happens to be in, say, a tumor suppressor gene, that would be a big concern.”

Potential for inefficiency, even harm

Although the study looked at CRISPR-Cas9 gene editing, the researchers believe their findings extend to other gene-editing tools such as zinc-finger nucleases (ZFN) and TAL effector nucleases.

“The unifying theme is that all these technologies rely on identifying stretches of DNA bases very specifically,” says Canver. “So, a variant that affects the target sequence could reduce guide RNA binding. Variants can also lead to binding at new sites that could potentially cause harm. As these gene-editing therapies continue to develop and start to approach the clinic, it’s important to make sure each therapy is going to be tailored to the patient that’s going to be treated.”


Samuel Lessard of the Montreal Heart Institute and Université de Montréal was the study’s first author. Co-authors were Laurent Francioli, Jessica Alfoldi, Patrick T. Ellinor and Daniel G. MacArthur of Massachusetts General Hospital and the Broad Institute of MIT and Harvard, and Jean-Claude Tardif and Guillaume Lettre of the Montreal Heart Institute and Université de Montréal. In addition to their Boston Children’s Hospital/Dana-Farber affiliations, Orkin and Canver are members of the Harvard Stem Cell Institute, and Orkin is also a Howard Hughes Medical Institute investigator.

The study was funded by Genome Canada and Genome Quebec, the Canada Research Chair Program, the MHI Foundation, the National Heart, Lung and Blood Institute (P01HL032262) and the National Institute of Diabetes and Digestive and Kidney Diseases (P30DK049216, F30DK103359).