Structure and Engineering of Francisella novicida Cas9

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 targetab...

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Bibliographic Details
Published in:Cell Vol. 164; no. 5; p. 950
Main Authors: Hirano, Hisato, Gootenberg, Jonathan S, Horii, Takuro, Abudayyeh, Omar O, Kimura, Mika, Hsu, Patrick D, Nakane, Takanori, Ishitani, Ryuichiro, Hatada, Izuho, Zhang, Feng, Nishimasu, Hiroshi, Nureki, Osamu
Format: Journal Article
Language:English
Published: United States 25.02.2016
Subjects:
Animals
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - metabolism
Blastocyst - metabolism
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Crystallography, X-Ray
Embryo, Mammalian - metabolism
Endonucleases - chemistry
Endonucleases - genetics
Endonucleases - metabolism
Francisella - enzymology
Genetic Engineering - methods
Mice
Microinjections - methods
Models, Molecular
RNA, Guide, CRISPR-Cas Systems - genetics
ISSN:1097-4172, 1097-4172
Online Access:Get more information
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Summary: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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ISSN:1097-4172
1097-4172
DOI:10.1016/j.cell.2016.01.039