An updated evolutionary classification of CRISPR–Cas systems including rare variants

Kira S. Makarova; Sergey A. Shmakov; Yuri I. Wolf; Pascal Mutz; Han Altae-Tran; Chase L. Beisel; Stan J. J. Brouns; Emmanuelle Charpentier; David Cheng; Jennifer Doudna; Daniel H. Haft; Philippe Horvath; Sylvain Moineau; Francisco J. M. Mojica; Patrick Pausch; Rafael Pinilla-Redondo; Shiraz A. Shah; Virginijus Siksnys; Michael P. Terns; Jesse Tordoff; Česlovas Venclovas; Malcolm F. White; Alexander F. Yakunin; Feng Zhang; Roger A. Garrett; Rolf Backofen; John van der Oost; Rodolphe Barrangou; Eugene V. Koonin

doi:10.1038/s41564-025-02180-8

An updated evolutionary classification of CRISPR–Cas systems including rare variants

Kira S. Makarova, Sergey A. Shmakov, Yuri I. Wolf, Pascal Mutz, Han Altae-Tran, Chase L. Beisel, Stan J. J. Brouns, Emmanuelle Charpentier, David Cheng, Jennifer Doudna, Daniel H. Haft, Philippe Horvath, Sylvain Moineau, Francisco J. M. Mojica, Patrick Pausch, Rafael Pinilla-Redondo, Shiraz A. Shah, Virginijus Siksnys, Michael P. Terns, Jesse TordoffČeslovas Venclovas, Malcolm F. White, Alexander F. Yakunin, Feng Zhang, Roger A. Garrett, Rolf Backofen, John van der Oost, Rodolphe Barrangou, Eugene V. Koonin^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

The known diversity of CRISPR–Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR–Cas systems. The updated CRISPR–Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR–Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR–Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR–Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.

Original language	English
Pages (from-to)	1-30
Number of pages	30
Journal	Nature Microbiology
DOIs	https://doi.org/10.1038/s41564-025-02180-8
Publication status	Published - 6 Nov 2025

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Makarova, K. S., Shmakov, S. A., Wolf, Y. I., Mutz, P., Altae-Tran, H., Beisel, C. L., Brouns, S. J. J., Charpentier, E., Cheng, D., Doudna, J., Haft, D. H., Horvath, P., Moineau, S., Mojica, F. J. M., Pausch, P., Pinilla-Redondo, R., Shah, S. A., Siksnys, V., Terns, M. P., ... Koonin, E. V. (2025). An updated evolutionary classification of CRISPR–Cas systems including rare variants. Nature Microbiology, 1-30. https://doi.org/10.1038/s41564-025-02180-8

@article{651cc3052bca4b3ca1ec042bb42f8bb7,

title = "An updated evolutionary classification of CRISPR–Cas systems including rare variants",

abstract = "The known diversity of CRISPR–Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR–Cas systems. The updated CRISPR–Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR–Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR–Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR–Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.",

author = "Makarova, {Kira S.} and Shmakov, {Sergey A.} and Wolf, {Yuri I.} and Pascal Mutz and Han Altae-Tran and Beisel, {Chase L.} and Brouns, {Stan J. J.} and Emmanuelle Charpentier and David Cheng and Jennifer Doudna and Haft, {Daniel H.} and Philippe Horvath and Sylvain Moineau and Mojica, {Francisco J. M.} and Patrick Pausch and Rafael Pinilla-Redondo and Shah, {Shiraz A.} and Virginijus Siksnys and Terns, {Michael P.} and Jesse Tordoff and {\v C}eslovas Venclovas and White, {Malcolm F.} and Yakunin, {Alexander F.} and Feng Zhang and Garrett, {Roger A.} and Rolf Backofen and {van der Oost}, John and Rodolphe Barrangou and Koonin, {Eugene V.}",

note = "Funding: K.S.M., S. A. Shmakov, Y.I.W., P.M., D.H.H. and E.V.K. are supported through the Intramural Research Program of the National Institutes of Health of the USA (National Library of Medicine). S. A. Shmakov is supported by funding of the Basic Research Program by the National Research University Higher School of Economics. C.L.B. is supported by European Research Council grants 865973 and 101158249. D.C. and J.T. are supported by Arbor Biotechnologies. P.H. is supported by IFF. S.M. was supported by funding from the Natural Sciences and Engineering Research Council of Canada (Discovery program) and holds a Tier 1 Canada Research Chair in Bacteriophages. F.J.M.M. is supported by the grants PID2023-150750NB-I00 (funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU), PROMETEU/2021/057 (funded by Conselleria d{\textquoteright}Educaci{\'o}, Cultura, Universitats i Ocupaci{\'o}, Generalitat Valenciana, Spain) and INNEST/2024/427 (funded by Agencia Valenciana de Innovaci{\'o}n—IVACE + I Innovaci{\'o}n—and by the European Union through the ERDF Program of the Valencian Community 2021–2027). P.P. receives funding from the EMBC under EMBO Installation Grant (5342-2023) and is supported by the LMT under the {\textquoteleft}University Excellence Initiatives{\textquoteright} (measure number 12-001-01-01-01, project S-A-UEI-23-10). R.P.-R. is supported by a Lundbeck Fonden grant (R347-2020-2346) and a research grant from VILLUM FONDEN (VIL60763). S. A. Shah is a recipient of a Novo Nordisk Foundation project grant in basic bioscience (NNF18OC0052965). C.V. is supported by intramural funds of the Vilnius University. M.P.T. is supported by the National Institutes of Health (grant R35GM118160). A.F.Y. is supported by an Environmental Biotechnology Innovation Centre (EBIC), UKRI Engineering Biology Mission Hub grant (BB/Y008332/1). F.Z. is supported by Howard Hughes Medical Institute; Yang Tan Collective; Broad Institute Programmable Therapeutics Gift Donors; Pershing Square Foundation, William Ackman, and Neri Oxman; Phillips family; J. and P. Poitras; B.T. Charitable Trust. R. Backofen is supported by the German Research Foundation (SFB 1597/1 {\textquoteleft}Small Data{\textquoteright}.",

year = "2025",

month = nov,

day = "6",

doi = "10.1038/s41564-025-02180-8",

language = "English",

pages = "1--30",

journal = "Nature Microbiology",

issn = "2058-5276",

publisher = "Nature Research",

}

Makarova, KS, Shmakov, SA, Wolf, YI, Mutz, P, Altae-Tran, H, Beisel, CL, Brouns, SJJ, Charpentier, E, Cheng, D, Doudna, J, Haft, DH, Horvath, P, Moineau, S, Mojica, FJM, Pausch, P, Pinilla-Redondo, R, Shah, SA, Siksnys, V, Terns, MP, Tordoff, J, Venclovas, Č, White, MF, Yakunin, AF, Zhang, F, Garrett, RA, Backofen, R, van der Oost, J, Barrangou, R & Koonin, EV 2025, 'An updated evolutionary classification of CRISPR–Cas systems including rare variants', Nature Microbiology, pp. 1-30. https://doi.org/10.1038/s41564-025-02180-8

TY - JOUR

T1 - An updated evolutionary classification of CRISPR–Cas systems including rare variants

AU - Makarova, Kira S.

AU - Shmakov, Sergey A.

AU - Wolf, Yuri I.

AU - Mutz, Pascal

AU - Altae-Tran, Han

AU - Beisel, Chase L.

AU - Brouns, Stan J. J.

AU - Charpentier, Emmanuelle

AU - Cheng, David

AU - Doudna, Jennifer

AU - Haft, Daniel H.

AU - Horvath, Philippe

AU - Moineau, Sylvain

AU - Mojica, Francisco J. M.

AU - Pausch, Patrick

AU - Pinilla-Redondo, Rafael

AU - Shah, Shiraz A.

AU - Siksnys, Virginijus

AU - Terns, Michael P.

AU - Tordoff, Jesse

AU - Venclovas, Česlovas

AU - White, Malcolm F.

AU - Yakunin, Alexander F.

AU - Zhang, Feng

AU - Garrett, Roger A.

AU - Backofen, Rolf

AU - van der Oost, John

AU - Barrangou, Rodolphe

AU - Koonin, Eugene V.

N1 - Funding: K.S.M., S. A. Shmakov, Y.I.W., P.M., D.H.H. and E.V.K. are supported through the Intramural Research Program of the National Institutes of Health of the USA (National Library of Medicine). S. A. Shmakov is supported by funding of the Basic Research Program by the National Research University Higher School of Economics. C.L.B. is supported by European Research Council grants 865973 and 101158249. D.C. and J.T. are supported by Arbor Biotechnologies. P.H. is supported by IFF. S.M. was supported by funding from the Natural Sciences and Engineering Research Council of Canada (Discovery program) and holds a Tier 1 Canada Research Chair in Bacteriophages. F.J.M.M. is supported by the grants PID2023-150750NB-I00 (funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU), PROMETEU/2021/057 (funded by Conselleria d’Educació, Cultura, Universitats i Ocupació, Generalitat Valenciana, Spain) and INNEST/2024/427 (funded by Agencia Valenciana de Innovación—IVACE + I Innovación—and by the European Union through the ERDF Program of the Valencian Community 2021–2027). P.P. receives funding from the EMBC under EMBO Installation Grant (5342-2023) and is supported by the LMT under the ‘University Excellence Initiatives’ (measure number 12-001-01-01-01, project S-A-UEI-23-10). R.P.-R. is supported by a Lundbeck Fonden grant (R347-2020-2346) and a research grant from VILLUM FONDEN (VIL60763). S. A. Shah is a recipient of a Novo Nordisk Foundation project grant in basic bioscience (NNF18OC0052965). C.V. is supported by intramural funds of the Vilnius University. M.P.T. is supported by the National Institutes of Health (grant R35GM118160). A.F.Y. is supported by an Environmental Biotechnology Innovation Centre (EBIC), UKRI Engineering Biology Mission Hub grant (BB/Y008332/1). F.Z. is supported by Howard Hughes Medical Institute; Yang Tan Collective; Broad Institute Programmable Therapeutics Gift Donors; Pershing Square Foundation, William Ackman, and Neri Oxman; Phillips family; J. and P. Poitras; B.T. Charitable Trust. R. Backofen is supported by the German Research Foundation (SFB 1597/1 ‘Small Data’.

PY - 2025/11/6

Y1 - 2025/11/6

N2 - The known diversity of CRISPR–Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR–Cas systems. The updated CRISPR–Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR–Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR–Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR–Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.

AB - The known diversity of CRISPR–Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR–Cas systems. The updated CRISPR–Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR–Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR–Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR–Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.

U2 - 10.1038/s41564-025-02180-8

DO - 10.1038/s41564-025-02180-8

M3 - Article

C2 - 41198952

AN - SCOPUS:105021238162

SN - 2058-5276

SP - 1

EP - 30

JO - Nature Microbiology

JF - Nature Microbiology

ER -

An updated evolutionary classification of CRISPR–Cas systems including rare variants

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