Cell

The RNA-guided DNA endonuclease Cas9 cleaves double-stranded DNA targets with a protospacer adjacent motif (PAM) and complementarity to the guide RNA. Recently, we harnessed Staphylococcus aureus Cas9 (SaCas9), which is significantly smaller than Streptococcus pyogenes Cas9 (SpCas9), to facilitate efficient in\uc2\ua0vivo genome editing. Here, we report the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets, containing the 5'-TTGAAT-3' PAM and the 5'-TTGGGT-3' PAM, at 2.6 and 2.7\uc2\ua0\uc3\u2026 resolutions, respectively. The structures revealed the mechanism of the relaxed recognition of the 5'-NNGRRT-3' PAM by SaCas9. A structural comparison of SaCas9 with SpCas9 highlighted both structural conservation and divergence, explaining their distinct PAM specificities and orthologous sgRNA recognition. Finally, we applied the structural information about this minimal Cas9 to rationally design compact transcriptional activators and inducible nucleases, to further expand the CRISPR-Cas9 genome editing toolbox.1DP1-MH100706/DP/NCCDPHP CDC HHS/United StatesDP1 MH100706/MH/NIMH NIH HHS/United StatesR01 MH110049/MH/NIMH NIH HHS/United StatesT32 GM007753/GM/NIGMS NIH HHS/United StatesT32 GM008313/GM/NIGMS NIH HHS/United StatesT32GM007753/GM/NIGMS NIH HHS/United States2016-08-27T00:00:00Z26317473PMC467026