Basis for discrimination by engineered CRISPR/Cas9 enzymes

CRISPR/Cas9 is a programmable genome editing tool that has been widely used for biological applications. While engineered Cas9s have been reported to increase discrimination against off-target cleavage compared with wild type Streptococcus pyogenes (SpCas9) in vivo, the mechanism for enhanced specificity has not been extensively characterized. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. While DNA unwinding is the rate-limiting step for on-target cleavage by SpCas9, we show that chemistry is seriously impaired by more than 100-fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the rate of chemistry without increasing the rate of DNA rewinding—the kinetic partitioning favors release rather than cleavage of a bound off-target substrate because chemistry is slow. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.