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CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases. A high-fidelity variant of Streptococcus pyogenes CRISPR-Cas9 is reported that lacks detectable off-target events as assessed by genome-wide break capture and targeted sequencing methods. High-precision gene editing The CRISPR-Cas9 nucleases now widely used in gene editing can be readily customized, but can also induce substantial genome-wide off-target mutations at sequences that resemble the on-target site. Keith Joung and colleagues report a high-fidelity variant of Cas9 from Streptococcus pyogenes that shows on-target activities comparable to the wild-type enzyme, but with off-target events that are undetectable by genome-wide break capture and targeted sequencing methods., Author(s): Benjamin P. Kleinstiver [sup.1] [sup.2] , Vikram Pattanayak [sup.1] [sup.2] , Michelle S. Prew [sup.1] , Shengdar Q. Tsai [sup.1] [sup.2] , Nhu T. Nguyen [sup.1] , Zongli Zheng [...]

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) (3-6). 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 Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities., CRISPR-Cas9 nucleases enable efficient genome editing in a wide variety of organisms and cell types (1,2). Target site recognition by Cas9 is programmed by a chimaeric single guide RNA (sgRNA) [...]