Structure and Engineering of Francisella novicida Cas9
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...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
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Elsevier
2017
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| Online Access: | http://hdl.handle.net/1721.1/112719 https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1826195241667919872 |
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| author | Hirano, Hisato Horii, Takuro Kimura, Mika Nakane, Takanori Ishitani, Ryuichiro Hatada, Izuho Nishimasu, Hiroshi Nureki, Osamu Gootenberg, Jonathan S Abudayyeh, Omar Osama Hsu, Patrick Zhang, Feng |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Hirano, Hisato Horii, Takuro Kimura, Mika Nakane, Takanori Ishitani, Ryuichiro Hatada, Izuho Nishimasu, Hiroshi Nureki, Osamu Gootenberg, Jonathan S Abudayyeh, Omar Osama Hsu, Patrick Zhang, Feng |
| author_sort | Hirano, Hisato |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T10:09:56Z |
| format | Article |
| id | mit-1721.1/112719 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T10:09:56Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1127192022-09-30T19:18:47Z Structure and Engineering of Francisella novicida Cas9 Hirano, Hisato Horii, Takuro Kimura, Mika Nakane, Takanori Ishitani, Ryuichiro Hatada, Izuho Nishimasu, Hiroshi Nureki, Osamu Gootenberg, Jonathan S Abudayyeh, Omar Osama Hsu, Patrick Zhang, Feng 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 Gootenberg, Jonathan S Abudayyeh, Omar Osama Hsu, Patrick Zhang, Feng 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. 2017-12-12T17:02:52Z 2017-12-12T17:02:52Z 2016-02 2016-01 2017-12-12T16:44:44Z Article http://purl.org/eprint/type/JournalArticle 0092-8674 1097-4172 http://hdl.handle.net/1721.1/112719 Hirano, Hisato et al. “Structure and Engineering of Francisella Novicida Cas9.” Cell 164, 5 (February 2016): 950–961 © 2016 Elsevier Inc https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 http://dx.doi.org/10.1016/j.cell.2016.01.039 Cell Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | Hirano, Hisato Horii, Takuro Kimura, Mika Nakane, Takanori Ishitani, Ryuichiro Hatada, Izuho Nishimasu, Hiroshi Nureki, Osamu Gootenberg, Jonathan S Abudayyeh, Omar Osama Hsu, Patrick Zhang, Feng Structure and Engineering of Francisella novicida Cas9 |
| title | Structure and Engineering of Francisella novicida Cas9 |
| title_full | Structure and Engineering of Francisella novicida Cas9 |
| title_fullStr | Structure and Engineering of Francisella novicida Cas9 |
| title_full_unstemmed | Structure and Engineering of Francisella novicida Cas9 |
| title_short | Structure and Engineering of Francisella novicida Cas9 |
| title_sort | structure and engineering of francisella novicida cas9 |
| url | http://hdl.handle.net/1721.1/112719 https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 |
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