金黄色葡萄球菌Cas9的晶体结构
Crystal Structure of Staphylococcus aureus Cas9.
作者信息
Nishimasu Hiroshi, Cong Le, Yan Winston X, Ran F Ann, Zetsche Bernd, Li Yinqing, Kurabayashi Arisa, Ishitani Ryuichiro, Zhang Feng, Nureki Osamu
机构信息
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Cell. 2015 Aug 27;162(5):1113-26. doi: 10.1016/j.cell.2015.08.007.
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.
RNA引导的DNA内切酶Cas9可切割具有前间隔序列邻近基序（PAM）且与引导RNA互补的双链DNA靶标。最近，我们利用了比化脓性链球菌Cas9（SpCas9）小得多的金黄色葡萄球菌Cas9（SaCas9），以促进高效的体内基因组编辑。在此，我们分别报道了与单引导RNA（sgRNA）及其双链DNA靶标（包含5'-TTGAAT-3' PAM和5'-TTGGGT-3' PAM）结合的SaCas9的晶体结构，分辨率分别为2.6 Å和2.7 Å。这些结构揭示了SaCas9对5'-NNGRRT-3' PAM的宽松识别机制。对SaCas9与SpCas9的结构比较突出了结构上的保守性和差异性，解释了它们不同的PAM特异性和直系同源sgRNA识别。最后，我们应用了关于这种最小Cas9的结构信息来合理设计紧凑的转录激活因子和诱导性核酸酶，以进一步扩展CRISPR-Cas9基因组编辑工具箱。
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