Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

Adler, B. A.; Al-Shimary, M. J.; Patel, J. R.; Ambruster, E.; Colognori, D.; Charles, E. J.; Miller, K. V.; Lahiri, A.; Trinidad, M.; Boger, R.; Nomburg, J.; Beurnier, S.; Cui, M. L.; Barrangou, R.; Mutalik, V. K.; Schoeniger, J. S.; Pogliano, J. A.; Savage, D. F.; Doudna, J. A.; Cress, B. F.

doi:10.1101/2023.09.18.558157

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bioRxiv · 10.1101/2023.09.18.558157

Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

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Abstract

Bacteriophages constitute one of the largest sources of unknown gene content in the biosphere. Even for well-studied model phages, robust experimental approaches to identify and study their essential genes remain elusive. We uncover and exploit the conserved vulnerability of the phage transcriptome to facilitate genome-wide protein expression knockdown via programmable RNA-binding protein dRfxCas13d (CRISPRi-ART) across diverse phages and their host. Establishing the first broad-spectrum phage functional genomics platform, we predict over 90 essential genes across four phage genomes, a third of which have no known function. These results highlight hidden infection strategies encoded in the most abundant biological entities on earth and provide a facile platform to study them.

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Adler, B. A., Al-Shimary, M. J., Patel, J. R., Ambruster, E., Colognori, D., Charles, E. J., Miller, K. V., Lahiri, A., Trinidad, M., Boger, R., Nomburg, J., Beurnier, S., Cui, M. L., Barrangou, R., Mutalik, V. K., Schoeniger, J. S., Pogliano, J. A., Savage, D. F., Doudna, J. A., Cress, B. F.. 2023-09-18. Genome-wide Characterization of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi. https://doi.org/10.1101/2023.09.18.558157

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