Target-dependent nickase activities of CRISPR-Cas nucleases Cpf1 and Cas9

Clustered regularly interspaced short palindromic repeat (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR-Cas nucleases Cpf1 (also known as Cas12a) and Cas9 exhibit differential guide RNA sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential guide RNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched dsDNA targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using guide RNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Sacchromyces cerevisae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology directed nicking.