1. Structure and Engineering of Francisella novicida Cas9.
- Author
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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, and Nureki, Osamu
- Subjects
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FRANCISELLA novicida , *ENDONUCLEASES , *GENOME editing , *NUCLEOPROTEINS , *LABORATORY mice , *MICROINJECTION (Cytology) - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2016
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