561. Staphyloccocus aureus Cas9: An Alternative Cas9 for Genome Editing Applications

Abstract

The CRISPR/Cas9 system that is broadly used for genome editing in myriad model organisms and cell lines is derived from the bacterial species Streptoccocus pyogenes. Here we report the use and characterization of another Type II CRISPR endonuclease Cas9, from the bacterium Staphylococcus aureus. This Cas9, encoded by ≈3.2kb of DNA, is substantially smaller than the S. pyogenes Cas9 yet delivers comparable DNA cleavage rates as measured by NHEJ-induced inserts and deletions in HEK293T cells. The S. aureus Cas9 has a Protospacer Adjacent Motif (PAM) that is well defined and different from the S. pyogenes Cas9 PAM, thus extending the range of specific target sites for any locus of interest. We characterize single guide RNAs specific to the S. aureus Cas9 with a range in the length of targeting sequences, investigating differences in on-target DNA cleavage efficiency as well as off-target activity. Off target activity assays include detection of cleavage events at chromosomally-integrated target sequences and at endogenous loci genome-wide. Taken together, our results show that the S. aureus Cas9 is an effective tool for genome editing with properties similar to but distinct from the more widely used S. pyogenes Cas9.