An engineered ScCas9 with broad PAM range and high specificity and activity

A hybrid CRISPR-Cas9 variant is able to specifically and efficiently target a larger proportion of the genome. CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9-Sc++ and a higher-fidelity mutant HiFi-Sc++-that have simultaneously broad 5 '-NNG-3 ' PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc++ and HiFi-Sc++ extend the use of CRISPR editing for diverse applications.