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Shortening guide RNA markedly improves specificity of CRISPR-Cas nucleases
Date:1/26/2014

A simple adjustment to a powerful gene-editing tool may be able to improve its specificity. In a report receiving advance online publication in Nature Biotechnology, Massachusetts General Hospital (MGH) investigators describe how adjusting the length of the the guide RNA (gRNA) component of the synthetic enzymes called CRISPR-Cas RNA-guided nucleases (RGNs) can substantially reduce the occurrence of DNA mutations at sites other than the intended target, a limitation the team had previously described just last year.

"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," says J. Keith Joung, MD, PhD, associate chief for Research in the MGH Department of Pathology and senior author of the report. "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 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.

"Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity," Joung explains. "That led us to wonder whether removing these nucleotides coul
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Contact: Sue McGreevey
smcgreevey@partners.org
617-724-2764
Massachusetts General Hospital
Source:Eurekalert

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