The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2017 Oct 7

PMID 28985502

Citations 91

Authors

Karthik Murugan

Kesavan Babu

Ramya Sundaresan

Rakhi Rajan

Dipali G Sashital

Affiliations

Soon will be listed here.

Abstract

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.

Citing Articles

Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex.

Liu Y, Wang L, Zhang Q, Fu P, Zhang L, Yu Y Nat Commun. 2025; 16(1):1335.

PMID: 39904990 PMC: 11794572. DOI: 10.1038/s41467-024-55716-7.

Applications of CRISPR Technologies in Forestry and Molecular Wood Biotechnology.

Cao H, Michels D, Vu G, Gailing O Int J Mol Sci. 2024; 25(21).

PMID: 39519342 PMC: 11547103. DOI: 10.3390/ijms252111792.

CRISPR-Cas13-mediated RNA editing in the silkworm .

Tang Y, Zhang X, Dai Z, Li H, Yang Y, Zhao T Zool Res. 2024; 45(6):1249-1260.

PMID: 39397244 PMC: 11668950. DOI: 10.24272/j.issn.2095-8137.2024.105.

Genome editing using type I-E CRISPR-Cas3 in mice and rat zygotes.

Yoshimi K, Kuno A, Yamauchi Y, Hattori K, Taniguchi H, Mikamo K Cell Rep Methods. 2024; 4(8):100833.

PMID: 39121862 PMC: 11384072. DOI: 10.1016/j.crmeth.2024.100833.

The CRISPR-Cas System and Clinical Applications of CRISPR-Based Gene Editing in Hematology with a Focus on Inherited Germline Predisposition to Hematologic Malignancies.

Kansal R Genes (Basel). 2024; 15(7).

PMID: 39062641 PMC: 11276294. DOI: 10.3390/genes15070863.

References

Kuscu C, Arslan S, Singh R, Thorpe J, Adli M . Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. Nat Biotechnol. 2014; 32(7):677-83. DOI: 10.1038/nbt.2916. View

Dagdas Y, Chen J, Sternberg S, Doudna J, Yildiz A . A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9. Sci Adv. 2017; 3(8):eaao0027. PMC: 5547770. DOI: 10.1126/sciadv.aao0027. View

Jinek M, Jiang F, Taylor D, Sternberg S, Kaya E, Ma E . Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science. 2014; 343(6176):1247997. PMC: 4184034. DOI: 10.1126/science.1247997. View

Sternberg S, Richter H, Charpentier E, Qimron U . Adaptation in CRISPR-Cas Systems. Mol Cell. 2016; 61(6):797-808. DOI: 10.1016/j.molcel.2016.01.030. View

Niewoehner O, Garcia-Doval C, Rostol J, Berk C, Schwede F, Bigler L . Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature. 2017; 548(7669):543-548. DOI: 10.1038/nature23467. View

Swarts D, Makarova K, Wang Y, Nakanishi K, Ketting R, Koonin E . The evolutionary journey of Argonaute proteins. Nat Struct Mol Biol. 2014; 21(9):743-53. PMC: 4691850. DOI: 10.1038/nsmb.2879. View

Sorek R, Lawrence C, Wiedenheft B . CRISPR-mediated adaptive immune systems in bacteria and archaea. Annu Rev Biochem. 2013; 82:237-66. DOI: 10.1146/annurev-biochem-072911-172315. View

Hille F, Charpentier E . CRISPR-Cas: biology, mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016; 371(1707). PMC: 5052741. DOI: 10.1098/rstb.2015.0496. View

Kim D, Kim J, Hur J, Been K, Yoon S, Kim J . Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016; 34(8):863-8. DOI: 10.1038/nbt.3609. View

10.

Wu D, Guan X, Zhu Y, Ren K, Huang Z . Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA. Cell Res. 2017; 27(5):705-708. PMC: 5520857. DOI: 10.1038/cr.2017.46. View

11.

Jackson R, Wiedenheft B . A Conserved Structural Chassis for Mounting Versatile CRISPR RNA-Guided Immune Responses. Mol Cell. 2015; 58(5):722-8. PMC: 4465340. DOI: 10.1016/j.molcel.2015.05.023. View

12.

Yang H, Gao P, Rajashankar K, Patel D . PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease. Cell. 2016; 167(7):1814-1828.e12. PMC: 5278635. DOI: 10.1016/j.cell.2016.11.053. View

13.

Zhong G, Wang H, Li Y, Tran M, Farzan M . Cpf1 proteins excise CRISPR RNAs from mRNA transcripts in mammalian cells. Nat Chem Biol. 2017; 13(8):839-841. PMC: 5577360. DOI: 10.1038/nchembio.2410. View

14.

Sternberg S, LaFrance B, Kaplan M, Doudna J . Conformational control of DNA target cleavage by CRISPR-Cas9. Nature. 2015; 527(7576):110-3. PMC: 4859810. DOI: 10.1038/nature15544. View

15.

Kleinstiver B, Pattanayak V, Prew M, Tsai S, Nguyen N, Zheng Z . High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016; 529(7587):490-5. PMC: 4851738. DOI: 10.1038/nature16526. View

16.

East-Seletsky A, OConnell M, Burstein D, Knott G, Doudna J . RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. Mol Cell. 2017; 66(3):373-383.e3. PMC: 5999320. DOI: 10.1016/j.molcel.2017.04.008. View

17.

Swarts D, van der Oost J, Jinek M . Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a. Mol Cell. 2017; 66(2):221-233.e4. PMC: 6879319. DOI: 10.1016/j.molcel.2017.03.016. View

18.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View

19.

Marraffini L . CRISPR-Cas immunity in prokaryotes. Nature. 2015; 526(7571):55-61. DOI: 10.1038/nature15386. View

20.

Zetsche B, Gootenberg J, Abudayyeh O, Slaymaker I, Makarova K, Essletzbichler P . Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015; 163(3):759-71. PMC: 4638220. DOI: 10.1016/j.cell.2015.09.038. View