Gene editing — one reagent doesn't fit all

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, warn researchers.

A study led by Boston Children’s Hospital and the University of Montreal 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 gene being targeted that would throw off the technology. Findings appear in the Proceedings of the National Academy of Sciences.

“Humans vary in their DNA sequences, and what is taken as the ‘normal’ DNA sequence for reference cannot account for all these differences,” said 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 maximise efficacy and minimise potential safety concerns.”

The research team, led by Orkin, Canver and Samuel Lessard of the University of Montreal, analysed 7444 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 3000 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 7444 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,” explained 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.”

Such occurrences in the genome turned out not to be uncommon. In fact, about 50% of the analysed 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.

“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,” said Canver. “If an off-target effect happens to be in, say, a tumour suppressor gene, that would be a big concern.”

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,” said 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.”

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 and the National Institute of Diabetes and Digestive and Kidney Diseases.