Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA

Abstract

Engineered high-fidelity Cas9s have increased discrimination against off-targets. Kinetic analyses of Cas9-HF1 and HypaCas9 engineered Cas9 variants show that their DNA cleavage is impaired by more than 100- fold, which leads to release rather than cleavage of a bound off-target substrate.