Cas9
生物
清脆的
引导RNA
基因组编辑
核糖核蛋白
DNA
核糖核酸
核酸内切酶
计算生物学
基因组工程
弗朗西塞拉
遗传学
反式激活crRNA
细胞生物学
基因
土拉弗朗西斯菌
毒力
作者
Hajime Hirano,Jonathan S. Gootenberg,Takuro Horii,Omar O. Abudayyeh,Masashi Kimura,Patrick Hsu,Takanori Nakane,Ryuichiro Ishitani,Izuho Hatada,Feng Zhang,Hiroshi Nishimasu,Osamu Nureki
出处
期刊:Cell
[Elsevier]
日期:2016-02-01
卷期号:164 (5): 950-961
被引量:286
标识
DOI:10.1016/j.cell.2016.01.039
摘要
The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.
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