Supplementary Data for 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.

Data S1. Plasmids used in this study.
Data S2. Bacterial strains used in this study.
Data S3. Bacteriophages used in this study.
Data S4. Oligonucleotides used in this study, not including oligo pools.
Data S5. Data table describing metadata, raw and normalized read counts, crRNA fitness values.
Data S6. Curated phage genome annotations for phages T4, T5, SUSP1, and PTXU04.
Data S7. Plaque size measurements for all plaque assays in this study.