Cas9 nucleases carry out strand-specific cleavage by using the conserved HNH and RuvC nuclease domains, which can be mutated and exploited for additional function37. An aspartate-to-alanine (D10A) mutation in the RuvC catalytic domain27,28 allows the Cas9 nickase mutant (Cas9n) to nick rather than cleave DNA to yield single-stranded breaks, and the subsequent preferential repair through HDR22 can potentially decrease the frequency of unwanted indel mutations from off-target DSBs. Appropriately offset sgRNA pairs can guide Cas9n to simultaneously nick both strands of the target locus to mediate a DSB, thus effectively increasing the specificity of target recognition43. In addition, a Cas9 mutant with both DNA-cleaving catalytic residues mutated has been adapted to enable transcriptional regulation in Escherichia coli44, demonstrating the potential of functionalizing Cas9 for diverse applications, such as recruitment of fluorescent protein labels or chromatin- modifying enzymes to specific genomic loci for reporting or modulating gene function.
