While gene editing using enzymes like CRISPR has shown promise as a way to treat genetic disease, a new study conducted by researchers at Boston Children’s Hospital and the University of Montreal suggests that clinical investigators may need to take a few things into account when recruiting patients for clinical trials. According to the research team – who published their findings in the journal PNAS – an individual’s genetics may affect how well gene editing works, potentially increasing the risk of so-called “off-target” effects.
By studying each patient’s genome to determine whether they carry similar stretches of DNA as the target sequence, researchers may be able to improve the safety and efficacy of gene editing. However, this additional step could be a barrier preventing widespread use of 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,” said Dr. Stuart Orkin, of Dana-Farber Boston Children’s Cancer and Blood Disorders Center “We recommend that common variation be taken into account in designing targeting systems for therapeutic editing, to maximize efficacy and minimize potential safety concerns.”
After analyzing nearly 750 whole-genome sequences, the researchers aimed to study the differences in disease-causing gene mutations. Orkin and his team narrowed their search to around 3,000 guide RNAs (gRNAs) designed to target about 30 genetic disease-causing mutations and fix them using CRSPR-Cas9 gene editing.
“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,” said Matthew Canver, an MD-PhD student at Harvard Medical School. “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.”
What the research team found was striking: around half of all the gRNAs analyzed in the study showed some level of variability between individuals. Even more concerning was the finding of similar DNA sequences in other parts of the genome which could increase the risk of off-target gene edits.
“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 tumor suppressor gene, that would be a big concern.”
While this study focused on CRISPR-Cas9 gene editing, other forms of this technology – such as zinc-finger nucleases (ZFN) and TAL effector nucleases – are also under investigation as potential therapeutics. The researchers content that the results from the current study could also be applied to these alternative types of gene editing.
“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.”