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

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.

A transcriptionally active lipid vesicle encloses the injectedChimalliviridaegenome in early infection

“…Therefore, to determine whether the DAPI-stained puncta were Goslar genomes arrested at an early stage of infection, we investigated whether sfGFP-ChmA colocalized with them. sfGFP-ChmA can be expressed in the knockdown strain because the target sequence is not at the translational start site of the fusion protein (12,13). We expressed sfGFP-ChmA at a low concentration from a second plasmid to avoid complementation of ChmA knockdown.…”

“…CRISPRi-ART (CRISPR interference by Antisense RNA Targeting) uses catalytically inactive Ruminococcus flavefaciens Cas13d (dRfxCas13d) to specifically suppress expression of proteins of interest (Fig. 1B) (12,13). Guide-directed binding of targeted RNA triggers collateral RNAse trans-activity in wildtype Cas13, leading to death or dormancy of bacterial cells (14)(15)(16)(17).…”

“…Guide-directed binding of targeted RNA triggers collateral RNAse trans-activity in wildtype Cas13, leading to death or dormancy of bacterial cells (14)(15)(16)(17). However, mutation of key active site residues in dRfxCas13d's two HEPN (higher eukaryote and prokaryote nucleotide-binding) domains allows it to bind target RNA without inducing nuclease activity (12,13,18). Therefore, dRfxCas13d can be used to inhibit expression of a protein by binding and thereby occluding the translational start site of the corresponding mRNA (12,13,19).…”

“…However, mutation of key active site residues in dRfxCas13d's two HEPN (higher eukaryote and prokaryote nucleotide-binding) domains allows it to bind target RNA without inducing nuclease activity (12,13,18). Therefore, dRfxCas13d can be used to inhibit expression of a protein by binding and thereby occluding the translational start site of the corresponding mRNA (12,13,19). CRISPRi-ART has been shown to be highly effective as a genetics tool for diverse phages, including Goslar (12).…”

“…Therefore, dRfxCas13d can be used to inhibit expression of a protein by binding and thereby occluding the translational start site of the corresponding mRNA (12,13,19). CRISPRi-ART has been shown to be highly effective as a genetics tool for diverse phages, including Goslar (12).…”