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Diversity and evolution of class 2 CRISPR–Cas systems

Key Points

  • CRISPR–Cas systems form two major classes that differ in the organization of their effector modules. The effector modules of class 2 systems consist of a single large protein, which makes them the best candidates for genome-editing tools.

  • Computational methods of microbial genome screening were developed for the comprehensive identification of class 2 CRISPR–cas loci. Using these approaches, six new subtypes of the class 2 system were discovered, which brings the total for this class to three types and 10 subtypes.

  • Type II and type V CRISPR–Cas effectors are homologues of TnpB proteins, which are a poorly characterized family of nucleases that are encoded by bacterial and archaeal transposons. The different subtypes of these two types seem to have evolved independently, through the integration of TnpB-encoding transposons near CRISPR arrays.

  • Type VI effectors are large proteins that contain two RNase domains of the higher eukaryotes and prokaryotes nucleotide-binding domain (HEPN) superfamily and that have been shown to, or are predicted to, specifically target RNA.

  • The diverse class 2 CRISPR–Cas systems that have been discovered provide opportunities for the construction of versatile genome-editing tools.

Abstract

Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR–Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR–Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR–Cas.

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Figure 1: The updated classification scheme for class 2 CRISPR–Cas systems.
Figure 2: The domain architecture of class 2 CRISPR effector proteins.
Figure 3: Phylogenies of the type V and type VI-B effectors.
Figure 4: Possible routes of evolution for class 2 CRISPR–Cas systems.
Figure 5: Functional diversity of the experimentally characterized class 2 CRISPR–Cas systems.

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Acknowledgements

S.S., K.S.M., Y.I.W. and E.V.K. are supported by intramural funds of the US Department of Health and Human Services (to the US National Library of Medicine). K.S. is supported by a US National Institutes of Health (NIH) grant (GM10407), a Russian Science Foundation grant (14-14-00988), and by the Skolkovo Institute of Science and Technology (Skoltech). F.Z. is supported by the NIH through the US National Institute of Mental Health (NIMH; grants 5DP1-MH100706 and 1R01-MH110049); the US National Science Foundation (NSF); the New York Stem Cell Foundation; the Allen Distinguished Investigator Program; through The Paul G. Allen Frontiers Group; the Simons and Vallee Foundations; the Howard Hughes Medical Institute; the Skoltech–MIT Next Generation Program; James and Patricia Poitras and the Poitras Center for Affective Disorders; R. Metcalfe; and D. Cheng. F.Z. is a New York Stem Cell Foundation–Robertson Investigator.

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Correspondence to Feng Zhang or Eugene V. Koonin.

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F.Z. is a co-founder of, and scientific adviser for, Editas Medicine and is a scientific adviser for Horizon Discovery.

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Supplementary Methods description. (PDF 158 kb)

Supplementary information S2 (box)

Supplementary Excel Files (PDF 100 kb)

Supplementary information S3 (figure)

Multiple alignment of representatives from five V-U families. (PDF 1628 kb)

Supplementary information S4 (figure)

Strength of purifying selection for Class 2 effector protein families. (PDF 90 kb)

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UPGMA dendrogram of protein family profile similarity (PDF 136 kb)

Supplementary information S6 (figure)

Membrane proteins associated with Cas13b genes (PDF 153 kb)

Glossary

CRISPR–Cas

An adaptive immune system in archaea and bacteria that functions by inserting fragments of foreign genomes into CRISPR arrays and using the transcripts of the resulting spacers as guide RNAs to detect and inactivate the cognate genetic elements.

Adaptation

The first phase of the CRISPR immune response, during which a piece of foreign DNA is inserted into a CRISPR array to become a spacer that is subsequently used as the template to produce the CRISPR RNA (crRNA).

CRISPR RNAs

(crRNAs). Small RNA molecules that consist of the RNA complement of a spacer and parts of the two adjacent repeats. crRNAs are produced by processing of the transcript of the entire CRISPR array (pre-crRNA); processing is mediated either by Cas proteins only (class 1, type V-A and type VI-A systems) or by an external RNase, such as bacterial RNase III, in conjunction with Cas proteins.

Interference

The final phase of the CRISPR immune response, during which the target DNA (or less commonly, RNA) is recognized by a CRISPR effector through the bound CRISPR RNA (crRNA) and cleaved by the effector nuclease or nucleases.

Effector

A complex of Cas proteins (in class 1 systems), or a single, large protein (in class 2 systems), that is involved in target recognition and inactivation, and, in most cases, in the processing of pre-CRISPR RNA (pre-crRNA).

Class 2 CRISPR–Cas systems

One of the two major divisions of CRISPR–Cas that is characterized by effector modules that consist of a single, large protein with endonuclease activity.

Trans-acting CRISPR RNA

(tracrRNA). An accessory RNA molecule that is partially complementary to CRISPR and is involved in pre-crRNA processing in type II and type V-B CRISPR–Cas interference.

Higher eukaryotes and prokaryotes nucleotide-binding domains

(HEPN domains). An early name that was given when the functions of the domains were not well characterized. An expansive superfamily of domains with RNase activity that are involved in various defence functions, in particular, type VI and some class 1 CRISPR–Cas interference.

TnpB proteins

A poorly characterized superfamily of transposon-encoded proteins that contain RuvC-like nuclease domains. TnpB proteins are the apparent evolutionary ancestors of type II and type V CRISPR–Cas effectors.

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Shmakov, S., Smargon, A., Scott, D. et al. Diversity and evolution of class 2 CRISPR–Cas systems. Nat Rev Microbiol 15, 169–182 (2017). https://doi.org/10.1038/nrmicro.2016.184

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