Structure and Engineering of Francisella novicida Cas9
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nihms791924.pdf
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Author(s)
Hirano, Hisato
Horii, Takuro
Kimura, Mika
Nakane, Takanori
Ishitani, Ryuichiro
Hatada, Izuho
Nishimasu, Hiroshi
Nureki, Osamu
Gootenberg, Jonathan S
Abudayyeh, Omar Osama
Date Issued
February 2016
Journal
Cell
Publisher
Elsevier
Citation
Hirano, Hisato et al. “Structure and Engineering of Francisella Novicida Cas9.” Cell 164, 5 (February 2016): 950–961 © 2016 Elsevier Inc
Version
Author's final manuscript
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences
McGovern Institute for Brain Research at MIT
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Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/j.cell.2016.01.039
