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Harvard Scientists Combine Strategies to Mitigate CRISPR Off-Target Effects

NEW YORK (GenomeWeb) – In an effort to cut down on unintended consequences of CRISPR/Cas9 gene editing, scientists from Harvard Medical School have combined two strategies known to reduce off-target enzyme activity.

Led by Nicolas Wyvekens and Shengdar Tsai, the researchers looked at the effects of combining dimerizing RNA-guided Fok1-dCas9 fusion nucleases (RFNs) with truncated guide RNAs (tru-RNAs). They published their results yesterday in Human Gene Therapy.

Both RFNs and tru-gRNAs have been shown to increase specificity and decrease off-target effects. RFNs use a dead Cas9 (dCas9) to bind to the DNA but not to cut, using the Fok1 nuclease instead. However, Fok1 can edit only when it dimerizes; by designing guide RNAs that flank the cutting site towards each 5' end, the RFN complex achieves extra specificity. It must recruit two Fok1-dCas9s to enable cleavage at each target.

Tru-gRNAs have also been shown to reduce off-target editing while "generally maintaining" on-target activity, the authors wrote. They found that tru-gRNAs with 19 base pairs of complementarity to the target site, instead of the full 20, gave the best mix of decreased off-target and sustained on-target activity. They showed that 19-basepair gRNAs had about 90 percent of the on-target activity of full-length gRNAs.

The scientists showed that using both Fok1-dCas9 fusions and tru-gRNAS resulted in additive performance. They characterized off-target activity by using a deep sequencing method called GUIDE-Seq to find double-stranded breaks at off-target sites. GUIDE-Seq was developed by Tsai and Keith Joung, also a co-author on the Human Gene Therapy paper.

"On average, coexpression of FokI-dCas9 with tru-gRNAs as compared with conventional full-length gRNAs reduces unwanted indel mutation frequencies by 40 percent," the authors wrote, noting that there were still two sites where they saw off-target activity.

"Our data show that tru-RFNs provide a useful and further improved tool for high-precision genome editing applications in human cells," they wrote.

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