期刊
CELL
卷 164, 期 5, 页码 950-961出版社
CELL PRESS
DOI: 10.1016/j.cell.2016.01.039
关键词
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资金
- D.O.E. Computational Science Graduate Fellowship
- Basic Science and Platform Technology Program for Innovative Biological Medicine from the Japan Agency for Medical Research and Development (AMED)
- NIH through NIMH [5DP1-MH100706, 1R01-MH110049]
- NIH through NIDDK [5R01DK097768-03]
- New York Stem Cell Foundation
- Vallee Foundation
- Simons, Paul G. Allen Family Foundation
- JST
- PRESTO
- JSPS KAKENHI [26291010, 15H01463]
- Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science, and Technology
- Grants-in-Aid for Scientific Research [26291010, 15H01463] Funding Source: KAKEN
The RNA-guided endonuclease Cas9 cleaves doublestranded 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 angstrom 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 themore relaxed 5'-YG-3' PAM. Furthermore, wedemonstrated 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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