Crystal Structure of Staphylococcus aureus Cas9.

Cell
Authors
Keywords
Abstract

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 vivo 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 Å 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.

Year of Publication
2015
Journal
Cell
Volume
162
Issue
5
Pages
1113-26
Date Published
2015 Aug 27
ISSN
1097-4172
URL
DOI
10.1016/j.cell.2015.08.007
PubMed ID
26317473
PubMed Central ID
PMC4670267
Links
Grant list
R01 MH110049 / MH / NIMH NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
T32 GM008313 / GM / NIGMS NIH HHS / United States
T32GM007753 / GM / NIGMS NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States