Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

Sergey Shmakov; Omar O Abudayyeh; Kira S Makarova; Yuri I Wolf; Jonathan S Gootenberg; Ekaterina Semenova; Leonid Minakhin; Julia Joung; Silvana Konermann; Konstantin Severinov; Feng Zhang; Eugene V Koonin

doi:10.1016/j.molcel.2015.10.008

Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

Mol Cell. 2015 Nov 5;60(3):385-97. doi: 10.1016/j.molcel.2015.10.008. Epub 2015 Oct 22.

Affiliations

¹ Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia; NCBI, NLM, NIH, Bethesda, MD 20894, USA.
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 Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
³ NCBI, NLM, NIH, Bethesda, MD 20894, USA.
⁴ Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
⁵ 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.
⁶ 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 Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: [email protected].
⁷ NCBI, NLM, NIH, Bethesda, MD 20894, USA. Electronic address: [email protected].

Abstract

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.

Keywords: CRISPR-Cas adaptive immunity; Cas9; Cpf1; HEPN domain; PAM; RNA-seq; RuvC-like endonuclease; computational discovery pipeline; crRNA; tracrRNA.

Publication types

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

MeSH terms

Bacteria* / genetics
Bacteria* / metabolism
Bacterial Proteins* / genetics
Bacterial Proteins* / metabolism
CRISPR-Cas Systems / physiology*
Evolution, Molecular*
Protein Structure, Tertiary
RNA, Bacterial* / genetics
RNA, Bacterial* / metabolism
Recombination, Genetic / physiology
Ribonucleases* / genetics
Ribonucleases* / metabolism

Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

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