Engineered CRISPR-Cas9 nucleases with altered PAM specificities

CRISPR-Cas9 nucleases are widely used for genome editing, but the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM); here the commonly used Streptococcus pyogenes Cas9 (SpCas9) is modified to recognize alternative PAM sequences, enabling robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9. SpCas9 variants with altered PAM specificities Although CRISPR–Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). Keith Joung and colleagues modify the commonly used Streptococcus pyogenes Cas9 (SpCas9) to recognize alternative PAM sequences and demonstrate robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9. They also identify and characterize another SpCas9 variant that shows better specificity in human cells. The engineering strategies developed for this work can extended to Cas9 proteins from other species to yield altered and improved PAM specificities. Although CRISPR-Cas9 nucleases are widely used for genome editing 1 , 2 , the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM) 3 , 4 , 5 , 6 . As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis 7 . In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphyloco