C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Omar O Abudayyeh; Jonathan S Gootenberg; Silvana Konermann; Julia Joung; Ian M Slaymaker; David B T Cox; Sergey Shmakov; Kira S Makarova; Ekaterina Semenova; Leonid Minakhin; Konstantin Severinov; Aviv Regev; Eric S Lander; Eugene V Koonin; Feng Zhang

doi:10.1126/science.aaf5573

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

Science. 2016 Aug 5;353(6299):aaf5573. doi: 10.1126/science.aaf5573. Epub 2016 Jun 2.

Authors

Affiliations

¹ Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
² Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
³ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁴ Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
⁵ Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia. National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
⁶ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
⁷ Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
⁸ Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia. Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA. Institute of Molecular Genetics, Russian Academy of Sciences, Moscow, 123182, Russia.
⁹ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
¹¹ National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. zhang@broadinstitute.org koonin@ncbi.nlm.nih.gov.
¹² Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA. Departments of Brain and Cognitive Science and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. zhang@broadinstitute.org koonin@ncbi.nlm.nih.gov.

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated genes (Cas) adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the class 2 type VI CRISPR-Cas effector C2c2 and demonstrate its RNA-guided ribonuclease function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis shows that C2c2 is guided by a single CRISPR RNA and can be programmed to cleave single-stranded RNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, mutations of which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Publication types

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

MeSH terms

Adaptive Immunity / genetics*
Bacterial Proteins / chemistry
Bacterial Proteins / genetics
Bacterial Proteins / metabolism*
Base Sequence
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats
Endoribonucleases / chemistry
Endoribonucleases / genetics
Endoribonucleases / metabolism*
Escherichia coli / genetics
Escherichia coli / immunology
Escherichia coli / virology
Gene Knockdown Techniques
Genetic Loci
Leptotrichia / enzymology*
Leptotrichia / genetics
Leptotrichia / immunology
Levivirus / immunology
Molecular Sequence Data
Protein Structure, Tertiary
RNA Cleavage
RNA, Guide, CRISPR-Cas Systems / metabolism*

Substances

Bacterial Proteins
RNA, Guide, CRISPR-Cas Systems
Endoribonucleases

Abstract

Publication types

MeSH terms

Substances

Grants and funding