Structure and Engineering of Francisella novicida Cas9

Cell. 2016 Feb 25;164(5):950-61. doi: 10.1016/j.cell.2016.01.039. Epub 2016 Feb 11.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Blastocyst / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Crystallography, X-Ray
  • Embryo, Mammalian / metabolism
  • Endonucleases / chemistry*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Francisella / enzymology*
  • Genetic Engineering / methods*
  • Mice
  • Microinjections / methods
  • Models, Molecular
  • RNA, Guide, CRISPR-Cas Systems / genetics

Substances

  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9
  • Cas9 protein, Francisella novicida
  • Endonucleases