Widespread CRISPR Repeat-like RNA Regulatory Elements in CRISPR-Cas Systems

Overview

Journal bioRxiv

Date 2023 Apr 24

PMID 37090614

Authors

Sergey A Shmakov

Zachary K Barth

Kira S Makarova

Yuri I Wolf

Vyacheslav Brover

Joseph E Peters

Eugene V Koonin

Affiliations

Soon will be listed here.

Abstract

CRISPR- loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of genes, in particular , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

References

Cheng F, Wu A, Liu C, Cao X, Wang R, Shu X . The toxin-antitoxin RNA guards of CRISPR-Cas evolved high specificity through repeat degeneration. Nucleic Acids Res. 2022; 50(16):9442-9452. PMC: 9458426. DOI: 10.1093/nar/gkac712. View

Nunez J, Bai L, Harrington L, Hinder T, Doudna J . CRISPR Immunological Memory Requires a Host Factor for Specificity. Mol Cell. 2016; 62(6):824-833. DOI: 10.1016/j.molcel.2016.04.027. View

Pawluk A, Davidson A, Maxwell K . Anti-CRISPR: discovery, mechanism and function. Nat Rev Microbiol. 2017; 16(1):12-17. DOI: 10.1038/nrmicro.2017.120. View

Yang S, Zhang Y, Xu J, Zhang J, Zhang J, Yang J . Orthogonal CRISPR-associated transposases for parallel and multiplexed chromosomal integration. Nucleic Acids Res. 2021; 49(17):10192-10202. PMC: 8464060. DOI: 10.1093/nar/gkab752. View

Makarova K, Wolf Y, Alkhnbashi O, Costa F, Shah S, Saunders S . An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015; 13(11):722-36. PMC: 5426118. DOI: 10.1038/nrmicro3569. View

Klompe S, Jaber N, Beh L, Mohabir J, Bernheim A, Sternberg S . Evolutionary and mechanistic diversity of Type I-F CRISPR-associated transposons. Mol Cell. 2022; 82(3):616-628.e5. PMC: 8849592. DOI: 10.1016/j.molcel.2021.12.021. View

Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire M, Geer L, Geer R . CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res. 2012; 41(Database issue):D348-52. PMC: 3531192. DOI: 10.1093/nar/gks1243. View

Chen Y, Liu J, Zhi S, Zheng Q, Ma W, Huang J . Repurposing type I-F CRISPR-Cas system as a transcriptional activation tool in human cells. Nat Commun. 2020; 11(1):3136. PMC: 7305327. DOI: 10.1038/s41467-020-16880-8. View

Sibley M, Raleigh E . A versatile element for gene addition in bacterial chromosomes. Nucleic Acids Res. 2011; 40(3):e19. PMC: 3273789. DOI: 10.1093/nar/gkr1085. View

10.

Koonin E, Makarova K . Evolutionary plasticity and functional versatility of CRISPR systems. PLoS Biol. 2022; 20(1):e3001481. PMC: 8730458. DOI: 10.1371/journal.pbio.3001481. View

11.

Santiago-Frangos A, Buyukyoruk M, Wiegand T, Krishna P, Wiedenheft B . Distribution and phasing of sequence motifs that facilitate CRISPR adaptation. Curr Biol. 2021; 31(16):3515-3524.e6. PMC: 8552246. DOI: 10.1016/j.cub.2021.05.068. View

12.

Soding J . Protein homology detection by HMM-HMM comparison. Bioinformatics. 2004; 21(7):951-60. DOI: 10.1093/bioinformatics/bti125. View

13.

Shmakov S, Wolf Y, Savitskaya E, Severinov K, Koonin E . Mapping CRISPR spaceromes reveals vast host-specific viromes of prokaryotes. Commun Biol. 2020; 3(1):321. PMC: 7308287. DOI: 10.1038/s42003-020-1014-1. View

14.

Ratner H, Escalera-Maurer A, Le Rhun A, Jaggavarapu S, Wozniak J, Crispell E . Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence. Mol Cell. 2019; 75(3):498-510.e5. PMC: 7205310. DOI: 10.1016/j.molcel.2019.05.029. View

15.

Workman R, Pammi T, Nguyen B, Graeff L, Smith E, Sebald S . A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression. Cell. 2021; 184(3):675-688.e19. DOI: 10.1016/j.cell.2020.12.017. View

16.

Barrangou R, Horvath P . A decade of discovery: CRISPR functions and applications. Nat Microbiol. 2017; 2:17092. DOI: 10.1038/nmicrobiol.2017.92. View

17.

Pons B, van Houte S, Westra E, Chevallereau A . Ecology and evolution of phages encoding anti-CRISPR proteins. J Mol Biol. 2023; 435(7):167974. DOI: 10.1016/j.jmb.2023.167974. View

18.

Faure G, Makarova K, Koonin E . CRISPR-Cas: Complex Functional Networks and Multiple Roles beyond Adaptive Immunity. J Mol Biol. 2018; 431(1):3-20. DOI: 10.1016/j.jmb.2018.08.030. View

19.

Yan W, Hunnewell P, Alfonse L, Carte J, Keston-Smith E, Sothiselvam S . Functionally diverse type V CRISPR-Cas systems. Science. 2018; 363(6422):88-91. PMC: 11258546. DOI: 10.1126/science.aav7271. View

20.

Rollins M, Chowdhury S, Carter J, Golden S, Miettinen H, Santiago-Frangos A . Structure Reveals a Mechanism of CRISPR-RNA-Guided Nuclease Recruitment and Anti-CRISPR Viral Mimicry. Mol Cell. 2019; 74(1):132-142.e5. PMC: 6521718. DOI: 10.1016/j.molcel.2019.02.001. View