Scientists at Massachusetts General Hospital (MGH) say a simple adjustment to a CRISPR may be able to improve its specificity. They describe in an advance online article in Nature Biotechnology how adjusting the length of the guide RNA (gRNA) component of CRISPR-Cas RNA-guided nucleases (RGNs) can substantially reduce the occurrence of DNA mutations at sites other than the intended target.
“Simply by shortening the length of the gRNA targeting region, we saw reductions in the frequencies of unwanted mutations at all of the previously known off-target sites we examined,” notes J. Keith Joung, M.D., Ph.D., associate chief for research in the department of pathology and senior author of the report (“Improving CRISPR-Cas nuclease specificity using truncated guide RNAs”). “Some sites showed decreases in mutation frequency of 5,000-fold or more, compared with full length gRNAs, and importantly these truncated gRNAs, which we call tru-gRNAs, are just as efficient as full-length gRNAs at reaching their intended target DNA segments.”
CRISPR-Cas RGNs combine a gene-cutting enzyme called Cas9 with a short RNA segment and are used to induce breaks in a complementary DNA segment in order to introduce genetic changes. Last year Dr. Joung’s team reported finding that, in human cells, CRISPR-Cas RGNs could also cause mutations in DNA sequences with differences of up to five nucleotides from the target, which could seriously limit the proteins' clinical usefulness. The team followed up those findings by investigating a hypothesis that could seem counterintuitive, that shortening the gRNA segment might reduce off-target mutations.
“[We] report that truncated gRNAs, with shorter regions of target complementarity <20 nucleotides in length, can decrease undesired mutagenesis at some off-target sites by 5,000-fold or more without sacrificing on-target genome editing efficiencies,” the team wrote. “In addition, use of truncated gRNAs can further reduce off-target effects induced by pairs of Cas9 variants that nick DNA (paired nickases).”
“While we don’t fully understand the mechanism by which tru-gRNAs reduce off-target effects, our hypothesis is that the original system might have more energy than it needs, enabling it to cleave even imperfectly matched sites,” explains Dr. Joung, who is an associate professor of pathology at Harvard Medical School and co-founder of Editas Medicine, a genome editing company.
Jennifer A. Doudna, Ph.D., from the University of California, Berkeley, and George Church, Ph.D., from Harvard Medical School, are also co-founders of the firm.