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.
Show 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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