Structure and Engineering of Francisella novicida Cas9

Hisato Hirano; Jonathan S Gootenberg; Takuro Horii; Omar O Abudayyeh; Mika Kimura; Patrick D Hsu; Takanori Nakane; Ryuichiro Ishitani; Izuho Hatada; Feng Zhang; Hiroshi Nishimasu; Osamu Nureki

doi:10.1016/j.cell.2016.01.039

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.

Authors

Affiliations

¹ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
³ Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan.
⁴ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁵ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan; JST, PRESTO, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address: [email protected].
⁶ Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Electronic address: [email protected].

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

Abstract

Publication types

MeSH terms

Substances

Grants and funding