Activation mechanism and molecular engineering of Staphylococcus aureus Cas9

Abstract Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Furthermore, the precise nuclease activation mechanism of SaCas9 remains elusive. Here, we rationally engineered a SaCas9 variant (eSaCas9-NNG) with an expanded target scope and reduced off-target activity. The eSaCas9-NNG induced indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice. We further determined the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for the improved specificity and illuminating notable differences in the activation mechanisms between the small SaCas9 and the larger SpCas9. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.