Functional Genomics of Diverse Bacteriophages Enabled by RNA-Binding CRISPRi

Bacteriophages harbor a large reservoir of genes of unknown function, but only a small fraction of these genes are known to be important during infection. To address the need for scalable experimental approaches to study phage gene fitness and function, we leveraged the RNA-targeting protein dCas13d to selectively interfere with protein translation and thereby measure phage gene fitness across the transcriptome. We find CRISPR Interference through Antisense RNA-Targeting (CRISPRi-ART) to be effective for diverse phage types including diverse ssRNA, ssDNA and dsDNA phages and nucleus-forming jumbo phages. Using CRISPRi-ART, we determined a conserved role of diverse rII homologs in subverting phage Lambda RexAB-mediated superinfection immunity. At transcriptome-wide scale, we also identified previously uncharacterized genes in four diverse phages that are critical for phage fitness, including a polyvalent protein and a homolog of the bacterial post-transcriptional regulator CsrA. This work establishes a broad-spectrum phage functional genomics platform and uncovers over 90 novel genes important for phage fitness.