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Structure and Engineering of Francisella novicida Cas9
Open accessAbstract
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 pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox.
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Metadata
- Authors
- Hirano, H., Gootenberg, J. S., Horii, T., Abudayyeh, O. O., Kimura, M., Hsu, P. D., Nakane, T., Ishitani, R., Hatada, I., Zhang, F., Nishimasu, H., & Nureki, O.
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2016-02
- Volume
- 164
- Issue
- 5
- Pages
- 950-961
- ISSN
- 0092-8674 (ISSN-L)
- OpenAlex
- W2268491398
- Cited by
- 365
- References
- 13
- FWCI
- 21.67
- Type
- journal article
- Language
- en
- Open access
- bronze
- Licence
- Funders
- D.O.E. Computational Science Graduate Fellowship
- Japan Agency for Medical Research and Development
- NIH through NIMH (5DP1-MH100706, 1R01-MH110049)
- NIDDK (5R01DK097768-03)
- New York Stem Cell
- Simons
- Paul G. Allen Family
- Vallee Foundations
- JST, PRESTO
- JSPS KAKENHI (26291010, 15H01463)
- Ministry of Education, Culture, Sports, Science, and Technology
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