Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems

By Sergey A. Shmakov, Omar Abudayyeh, Kira S. Makarova, Yuri I. Wolf, Jonathan Gootenberg, E. Semenova, L. Minakhin, J. Keith Joung, Silvana Konermann, Konstantin Severinov and 2 more. Molecules and Cells, 2015

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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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/73f6f23491c9dfb545834dcf3e57e93c71d25b4b

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Citations in the corpus

Crystal structure of Cpf1 in complex with guide RNA and target DNA (2016, background): “…class 2 effectors, as well as the transposon-encoded TnpB proteins, which appear to be the evolutionary ancestors of the RuvC domains in the type II and type V effectors (Shmakov et al., 2015), are expected to shed further light on the evolution of this remarkable class of RNA-guided endonucleases.”
Biology and Applications of CRISPR Systems: Harnessing Nature's Toolbox for Genome Engineering. (2016, neutral): “Type V systems also contain a Cas9-like single nuclease, either Cpf1, C2c1, or C2c3, depending on the subtype (Shmakov et al., 2015; Zetsche et al., 2015a).”
RNA Editing with CRISPR-Cas13 (2017, background): “Cas13 enzymes have two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) endoRNase domains that mediate precise RNA cleavage with a preference for targets with protospacer flanking site (PFS) motifs observed biochemically and in bacteria (10, 11).”
Transcriptome engineering with RNA-targeting Type VI-D CRISPR effectors (2018, neutral): “…et al., 2017; Samai et al., 2015), type VI systems have been recently described as the only known single-effector CRISPR nucleases that exclusively target RNA (Abudayyeh et al., 2016; East-Seletsky et al., 2016; Konermann et al., 2018; Shmakov et al., 2015; Smargon et al., 2017; Yan et al., 2018).”
CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins (2018, background): “A community effort resulted in the classification of CRISPR-Cas systems into two classes, six types and 22 subtypes, according to their Cas proteins (2,3).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “Types I–III are the most studied, while types IV–VI were more recently identified (Makarova & Koonin, 2015; Makarova et al., 2015; Shmakov et al., 2015).”
Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy (2020, neutral): “To date, four type VI CRISPR-Cas13 subtypes have been identified: Cas13a (previously known as C2c2) (Abudayyeh et al., 2016; East-Seletsky et al., 2016; Shmakov et al., 2015), Cas13b (Smargon et al.”
CRISPR/Cas Systems towards Next-Generation Biosensing. (2019, background): “Cas13 Effector-based CRISPR/Cas Biosensing Systems Within the CRISPR/Cas family, Cas13a (previously called C2c2) is an RNA-guided RNase belonging to the type VI CRISPR/Cas system [30].”