Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.

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http://dx.doi.org/10.1002/1873-3468.14608DOI Listing

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