Molecular engineering and dynamic activation mechanism of a PAM-flexible compact CRISPR-Cas9

Staphylococcus aureus Cas9 (SaCas9), which is smaller than the widely-used Streptococcus pyogenes Cas9 (SpCas9), has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires an 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. In addition, the 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. 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 four sequential states, PAM-checking state, DNA-unwinding state, pre-catalytic state and catalytically active state, which illuminate notable differences in the activation mechanisms between small SaCas9 and 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.