title: Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.
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.
authors.affiliation: Skolkovo Institute of Science and Technology, Skolkovo, 143025, Russia; NCBI, NLM, NIH, Bethesda, MD 20894, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Shmakov
authors.affiliation: 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.
authors.forename: Omar O
authors.initials: OO
authors.lastname: Abudayyeh
authors.affiliation: NCBI, NLM, NIH, Bethesda, MD 20894, USA.
authors.forename: Kira S
authors.initials: KS
authors.lastname: Makarova
authors.affiliation: NCBI, NLM, NIH, Bethesda, MD 20894, USA.
authors.forename: Yuri I
authors.initials: YI
authors.lastname: Wolf
authors.affiliation: 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.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Gootenberg
authors.affiliation: Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Semenova
authors.affiliation: Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Leonid
authors.initials: L
authors.lastname: Minakhin
authors.affiliation: 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.
authors.forename: Julia
authors.initials: J
authors.lastname: Joung
authors.affiliation: 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.
authors.forename: Silvana
authors.initials: S
authors.lastname: Konermann
authors.affiliation: 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.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
authors.affiliation: 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: zhang@broadinstitute.org.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
authors.affiliation: NCBI, NLM, NIH, Bethesda, MD 20894, USA. Electronic address: koonin@ncbi.nlm.nih.gov.
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000502:physiology*; D019143:Evolution, Molecular; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic / Q000502:physiology; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012260:Ribonucleases
keywords: CRISPR-Cas adaptive immunity; Cas9; Cpf1; HEPN domain; PAM; RNA-seq; RuvC-like endonuclease; computational discovery pipeline; crRNA; tracrRNA