Crystal Structure of Staphylococcus aureus Cas9.
Crystal Structure of Staphylococcus aureus Cas9.
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DOI：
10.1016/j.cell.2015.08.007
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发表时间：
2015-08-27
期刊：
影响因子：
64.5
通讯作者：
Nureki O
中科院分区：
文献类型：
--
作者：
Nishimasu H;Cong L;Yan WX;Ran FA;Zetsche B;Li Y;Kurabayashi A;Ishitani R;Zhang F;Nureki O
RNA引导的DNA内切核酸酶Cas9切割具有前间区序列邻近基序（PAM）和与引导RNA互补的双链DNA靶标。最近，我们利用金黄色葡萄球菌Cas9（SaCas 9），它比化脓性链球菌Cas9（SpCas 9）小得多，以促进有效的体内基因组编辑。在这里，我们报告了SaCas 9与单向导RNA（sgRNA）及其双链DNA靶标（含有5′-TTGAAT-3′ PAM和5′-TTGGGT-3′ PAM）复合的晶体结构，分辨率分别为2.6和2.7 μ m。这些结构揭示了SaCas 9对5′-NNGRRT-3′ PAM的弛豫识别机制。SaCas 9与SpCas 9的结构比较突出了结构保守性和差异性，解释了它们不同的PAM特异性和正交sgRNA识别。最后，我们将这个最小化的Cas9的结构信息应用于合理设计紧凑的转录激活因子和诱导型核酸酶，进一步扩展CRISPR-Cas9基因组编辑工具箱。
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.