Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence

Overview

Journal Mol Cell

Publisher Cell Press

Specialty Cell Biology

Date 2019 Jul 2

PMID 31256988

Citations 35

Authors

Hannah K Ratner

Andres Escalera-Maurer

Anais Le Rhun

Siddharth Jaggavarapu

Jessie E Wozniak

Emily K Crispell

Emmanuelle Charpentier

David S Weiss

Affiliations

Soon will be listed here.

Abstract

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

Citing Articles

CRISPR-repressed toxin-antitoxin provides herd immunity against anti-CRISPR elements.

Shu X, Wang R, Li Z, Xue Q, Wang J, Liu J Nat Chem Biol. 2024; 21(3):337-347.

PMID: 39075253 DOI: 10.1038/s41589-024-01693-3.

A dynamic subpopulation of CRISPR-Cas overexpressers allows Streptococcus pyogenes to rapidly respond to phage.

Stoltzfus M, Workman R, Keith N, Modell J Nat Microbiol. 2024; 9(9):2410-2421.

PMID: 38997519 DOI: 10.1038/s41564-024-01748-0.

TnpB homologues exapted from transposons are RNA-guided transcription factors.

Wiegand T, Hoffmann F, Walker M, Tang S, Richard E, Le H Nature. 2024; 631(8020):439-448.

PMID: 38926585 PMC: 11702177. DOI: 10.1038/s41586-024-07598-4.

Emergence of RNA-guided transcription factors via domestication of transposon-encoded TnpB nucleases.

Wiegand T, Hoffmann F, Walker M, Tang S, Richard E, Le H bioRxiv. 2023; .

PMID: 38076855 PMC: 10705468. DOI: 10.1101/2023.11.30.569447.

Interrogating two extensively self-targeting Type I CRISPR-Cas systems in Xanthomonas albilineans reveals distinct anti-CRISPR proteins that block DNA degradation.

Wimmer F, Englert F, Wandera K, Alkhnbashi O, Collins S, Backofen R Nucleic Acids Res. 2023; 52(2):769-783.

PMID: 38015466 PMC: 10810201. DOI: 10.1093/nar/gkad1097.

References

Anders S, Pyl P, Huber W . HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014; 31(2):166-9. PMC: 4287950. DOI: 10.1093/bioinformatics/btu638. View

Dugar G, Leenay R, Eisenbart S, Bischler T, Aul B, Beisel C . CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9. Mol Cell. 2018; 69(5):893-905.e7. PMC: 5859949. DOI: 10.1016/j.molcel.2018.01.032. View

Strutt S, Torrez R, Kaya E, Negrete O, Doudna J . RNA-dependent RNA targeting by CRISPR-Cas9. Elife. 2018; 7. PMC: 5796797. DOI: 10.7554/eLife.32724. View

OConnell M, Oakes B, Sternberg S, East-Seletsky A, Kaplan M, Doudna J . Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature. 2014; 516(7530):263-6. PMC: 4268322. DOI: 10.1038/nature13769. View

Pourcel C, Salvignol G, Vergnaud G . CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology (Reading). 2005; 151(Pt 3):653-663. DOI: 10.1099/mic.0.27437-0. View

Marraffini L, Sontheimer E . CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science. 2008; 322(5909):1843-5. PMC: 2695655. DOI: 10.1126/science.1165771. View

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S . CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315(5819):1709-12. DOI: 10.1126/science.1138140. View

Westra E, Buckling A, Fineran P . CRISPR-Cas systems: beyond adaptive immunity. Nat Rev Microbiol. 2014; 12(5):317-26. DOI: 10.1038/nrmicro3241. View

Louwen R, Staals R, Endtz H, van Baarlen P, van der Oost J . The role of CRISPR-Cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev. 2014; 78(1):74-88. PMC: 3957734. DOI: 10.1128/MMBR.00039-13. View

10.

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Soria E . Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol. 2005; 60(2):174-82. DOI: 10.1007/s00239-004-0046-3. View

11.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337(6096):816-21. PMC: 6286148. DOI: 10.1126/science.1225829. View

12.

Jones C, Sampson T, Nakaya H, Pulendran B, Weiss D . Repression of bacterial lipoprotein production by Francisella novicida facilitates evasion of innate immune recognition. Cell Microbiol. 2012; 14(10):1531-43. PMC: 3443312. DOI: 10.1111/j.1462-5822.2012.01816.x. View

13.

Marraffini L, Sontheimer E . Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature. 2010; 463(7280):568-71. PMC: 2813891. DOI: 10.1038/nature08703. View

14.

Deltcheva E, Chylinski K, Sharma C, Gonzales K, Chao Y, Pirzada Z . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011; 471(7340):602-7. PMC: 3070239. DOI: 10.1038/nature09886. View

15.

Sampson T, Saroj S, Llewellyn A, Tzeng Y, Weiss D . A CRISPR/Cas system mediates bacterial innate immune evasion and virulence. Nature. 2013; 497(7448):254-7. PMC: 3651764. DOI: 10.1038/nature12048. View

16.

Bryksin A, Matsumura I . Rational design of a plasmid origin that replicates efficiently in both gram-positive and gram-negative bacteria. PLoS One. 2010; 5(10):e13244. PMC: 2951906. DOI: 10.1371/journal.pone.0013244. View

17.

Ma K, Cao Q, Luo S, Wang Z, Liu G, Lu C . Enhances Bacterial Virulence by Repressing the Transcriptional Regulator in Streptococcus agalactiae. Infect Immun. 2017; 86(3). PMC: 5820942. DOI: 10.1128/IAI.00552-17. View

18.

Anders C, Niewoehner O, Duerst A, Jinek M . Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014; 513(7519):569-73. PMC: 4176945. DOI: 10.1038/nature13579. View

19.

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

20.

Weiss D, Brotcke A, Henry T, Margolis J, Chan K, Monack D . In vivo negative selection screen identifies genes required for Francisella virulence. Proc Natl Acad Sci U S A. 2007; 104(14):6037-42. PMC: 1832217. DOI: 10.1073/pnas.0609675104. View