Guide-free Cas9 from Pathogenic Bacteria Causes Severe Damage to DNA

Overview

Journal Sci Adv

Specialties Biology
Science

Date 2020 Jun 30

PMID 32596446

Citations 21

Authors

Chinmoy Saha

Prarthana Mohanraju

Andrew Stubbs

Gaurav Dugar

Youri Hoogstrate

Gert-Jan Kremers

Wiggert A van Cappellen

Deborah Horst-Kreft

Charlie Laffeber

Joyce H G Lebbink

Serena Bruens

Duncan Gaskin

Dior Beerens

Maarten Klunder

Rob Joosten

Jeroen A A Demmers

Dik van Gent

Johan W Mouton

Peter J van der Spek

John van der Oost

Peter van Baarlen

Rogier Louwen

Affiliations

Soon will be listed here.

Abstract

CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

Citing Articles

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References

Lara-Tejero M, Galan J . A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein. Science. 2000; 290(5490):354-7. DOI: 10.1126/science.290.5490.354. View

Ihry R, Worringer K, Salick M, Frias E, Ho D, Theriault K . p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nat Med. 2018; 24(7):939-946. DOI: 10.1038/s41591-018-0050-6. View

Gilbert M, Godschalk P, Karwaski M, Ang C, van Belkum A, Li J . Evidence for acquisition of the lipooligosaccharide biosynthesis locus in Campylobacter jejuni GB11, a strain isolated from a patient with Guillain-Barré syndrome, by horizontal exchange. Infect Immun. 2004; 72(2):1162-5. PMC: 321600. DOI: 10.1128/IAI.72.2.1162-1165.2004. View

Louwen R, Nieuwenhuis E, van Marrewijk L, Horst-Kreft D, de Ruiter L, Heikema A . Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures. Infect Immun. 2012; 80(9):3307-18. PMC: 3418735. DOI: 10.1128/IAI.06270-11. View

Talukder K, Aslam M, Islam Z, Azmi I, Dutta D, Hossain S . Prevalence of virulence genes and cytolethal distending toxin production in Campylobacter jejuni isolates from diarrheal patients in Bangladesh. J Clin Microbiol. 2008; 46(4):1485-8. PMC: 2292937. DOI: 10.1128/JCM.01912-07. View

Mortensen N, Schiellerup P, Boisen N, Klein B, Locht H, AbuOun M . The role of Campylobacter jejuni cytolethal distending toxin in gastroenteritis: toxin detection, antibody production, and clinical outcome. APMIS. 2011; 119(9):626-634. DOI: 10.1111/j.1600-0463.2011.02781.x. View

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T . Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7):676-82. PMC: 3855844. DOI: 10.1038/nmeth.2019. 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

Sundaresan R, Parameshwaran H, Yogesha S, Keilbarth M, Rajan R . RNA-Independent DNA Cleavage Activities of Cas9 and Cas12a. Cell Rep. 2017; 21(13):3728-3739. PMC: 5760271. DOI: 10.1016/j.celrep.2017.11.100. View

10.

Fonfara I, Le Rhun A, Chylinski K, Makarova K, Lecrivain A, Bzdrenga J . Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 2013; 42(4):2577-90. PMC: 3936727. DOI: 10.1093/nar/gkt1074. View

11.

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

12.

Louwen R, Horst-Kreft D, De Boer A, van der Graaf L, de Knegt G, Hamersma M . A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome. Eur J Clin Microbiol Infect Dis. 2012; 32(2):207-26. DOI: 10.1007/s10096-012-1733-4. View

13.

Jang K, Sweredoski M, Graham R, Hess S, Clemons Jr W . Comprehensive proteomic profiling of outer membrane vesicles from Campylobacter jejuni. J Proteomics. 2014; 98:90-8. PMC: 4534003. DOI: 10.1016/j.jprot.2013.12.014. View

14.

Dugar G, Svensson S, Bischler T, Waldchen S, Reinhardt R, Sauer M . The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni. Nat Commun. 2016; 7:11667. PMC: 4894983. DOI: 10.1038/ncomms11667. View

15.

Mohanraju P, Makarova K, Zetsche B, Zhang F, Koonin E, van der Oost J . Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems. Science. 2016; 353(6299):aad5147. DOI: 10.1126/science.aad5147. View

16.

Crosetto N, Mitra A, Silva M, Bienko M, Dojer N, Wang Q . Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat Methods. 2013; 10(4):361-5. PMC: 3651036. DOI: 10.1038/nmeth.2408. View

17.

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

18.

Nielsen H, Persson S, Olsen K, Ejlertsen T, Kristensen B, Schonheyder H . Bacteraemia with Campylobacter jejuni: no association with the virulence genes iam, cdtB, capA or virB. Eur J Clin Microbiol Infect Dis. 2010; 29(3):357-8. DOI: 10.1007/s10096-009-0863-9. View

19.

Mohanraju P, van der Oost J, Jinek M, Swarts D . Heterologous Expression and Purification of CRISPR-Cas12a/Cpf1. Bio Protoc. 2021; 8(9):e2842. PMC: 8275269. DOI: 10.21769/BioProtoc.2842. View

20.

Skarp C, Akinrinade O, Nilsson A, Ellstrom P, Myllykangas S, Rautelin H . Comparative genomics and genome biology of invasive Campylobacter jejuni. Sci Rep. 2015; 5:17300. PMC: 4658567. DOI: 10.1038/srep17300. View