Homo sapiens isolate:embryo kideny | cultivar:van der Eb''s lab Targeted Locus (Loci)

The use of paired Cas9 nickases instead of Cas9 nuclease drastically reduce off-target effects. Because both nickases must function for a nickase pair to make a double-strand break, the efficiency of paired nickases can intuitively be expected to be comparable or slightly lower than that of either corresponding nuclease alone. Here we carefully compared the gene-disrupting efficiency of Cas9 paired nickases with that of nucleases. Counterintuitively, the on-target efficiency of paired nickases is frequently higher than that of either corresponding. As the underlying mechanisms, we found that HNH domain preserved in D10A Cas9 nickase has a higher cleavage efficiency than RuvC domain. In addition, we show that the flow cytometric enrichment of cells containing paired nickase-induced mutations results in a population that lacks detectable off-target effects, enabling efficient knockout clone generation. We envision that our findings will greatly facilitate the use of paired Cas9 nickases.