Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA

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 OostPeter Van Baarlen, Rogier Louwen

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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, Campylobacter jejuni 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 Streptococcus pyogenes (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.

Original languageEnglish
Article numbereaaz4849
JournalScience Advances
Volume6
Issue number25
DOIs
Publication statusPublished - Jun 2020

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    Saha, C., Mohanraju, P., Stubbs, A., Dugar, G., Hoogstrate, Y., Kremers, G. J., Van Cappellen, W. A., Horst-Kreft, D., Laffeber, C., Lebbink, J. H. G., Bruens, S., Gaskin, D., Beerens, D., Klunder, M., Joosten, R., Demmers, J. A. A., Van Gent, D., Mouton, J. W., Van Der Spek, P. J., ... Louwen, R. (2020). Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA. Science Advances, 6(25), [eaaz4849]. https://doi.org/10.1126/sciadv.aaz4849