Discovery and engineering of small SlugCas9 with broad targeting range and high specificity and activity

The compact CRISPR/Cas9 system, which can be delivered by adeno-associated virus (AAV), is a promising platform for therapeutic applications. However, current compact Cas9 nucleases have limited activity, targeting scope and specificity. Here, we identified three compact SaCas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Interestingly, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. We further generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We lastly engineered SlugCas9 with mutations to generate a high fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.