PURedit Cas9 – A high specificity and high efficiency genome editing RNP system developed through protein engineering and RNP enhancer screening

Streptococcus pyogenes CRISPR Cas9 (SpCas9) has become an important tool for genome editing. To alleviate the off-target cleavage by Cas9, various SpCas9 variants with improved specificity have been developed through structure-based rational design or directed evolution. However, these specificity enhanced variants have lower nuclease activity, resulting in a substantial reduction in editing efficiency, especially when they are delivered in the form of pre-assembled ribonucleoprotein (RNP) complexes. To minimize such conflict between specificity and activity, we utilized a different engineering strategy by mutating a wide range of critical residues that are hypothetically involved in different DNA binding modalities and evaluating these mutants in human cells in the form of a ribonuclear protein complex of Cas9 with synthetic single guide RNA (sgRNA). We found that amino acid residue substitution with structurally somewhat similar residues can better retain nuclease activity while improving specificity, as compared with the widely used alanine substitution in prior engineering efforts. By stratifying mutant proteins on a set of target and off-target sites, we identified several mutants that possess balanced specificity and activity. Furthermore, to ensure high editing efficiency for these mutant proteins even on difficult-to-edit sites, we also developed an RNP enhancer, which can substantially increase Cas9 nuclease editing efficiency.. By combining one of the high specificity mutant proteins with the RNP enhancer, we developed a PURedit Cas9 RNP system, which exceeds commercially available wildtype and high fidelity Cas9 proteins in both activity and specificity on diverse endogenous sites.