Structure and Engineering of Francisella novicida Cas9

Details

• Alternative Title:
  Cell
• Personal Author:
  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
• Description:
  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.
• Subjects:
  Animals Bacterial Proteins Blastocyst CRISPR-Associated Protein 9 CRISPR-Cas Systems Crystallography, X-Ray Embryo, Mammalian Endonucleases Francisella Genetic Engineering Mice Microinjections Models, Molecular RNA, Guide, CRISPR-Cas Systems

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• Source:
  Cell. 164(5):950-961.
• Pubmed ID:
  26875867
• Pubmed Central ID:
  PMC4899972
• Document Type:
  Journal Article
• Funding:
  1R01-MH110049/MH/NIMH NIH HHS/United States ; 5DP1-MH100706/DP/NCCDPHP CDC HHS/United States ; 5R01DK097768-03/DK/NIDDK NIH HHS/United States ; DP1 MH100706/MH/NIMH NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States
• Volume:
  164
• Issue:
  5
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:618e1c8ef44475d2157e1c842f7439dd60f22f59bf1bf131db199e91f50feed4
• Download URL:
  https://stacks.cdc.gov/view/cdc/40093/cdc_40093_DS1.pdf
• File Type:
  [PDF - 3.62 MB ]