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

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. ### Competing Interest Statement The Regents of the University of California have patents pending related to this work on which B.A.A., M.J.A., J.R.P., E.A., E.J.C., J.A.P., D.F.S., J.A.D., and B.F.C. are inventors. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics and Mammoth Biosciences, a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Mammoth Biosciences and Inari, and is a Director at Johnson & Johnson and has sponsored research projects by Biogen, Roche and Pfizer. V.K.M. is a co-founder of Felix Biotechnology. R.B. is a shareholder of Caribou Biosciences, Intellia Therapeutics, Locus Biosciences, Inari, TreeCo, and Ancilia Biosciences.