Genome-wide CRISPRi (EcoWG1) screen of the P2 Old anti-phage defence system in Escherichia coli (pFD266)
Pooled genome-wide CRISPR interference (CRISPRi) screen in Escherichia coli K-12 MG1655 carrying the P2 Old (Old) anti-phage defence system on plasmid pFD266. The EcoWG1 dCas9 sgRNA library was introduced and guide abundance quantified by amplicon sequencing at the input timepoint (T0) and after DAPG induction of dCas9 (T3), in two biological replicates each, alongside an sfGFP-expressing control (pFR66, in place of the defence system) and the conjugation-donor input library (E. coli MFDpir, pFR56). Read 1 (22 sequencing cycles, custom Read 1 primer) carries the 20 bp sgRNA guide and read 2 is a 12 bp unique molecular identifier (umi1+umi2). Comparing guide abundance identifies host genes whose knockdown modulates P2 Old activity.
Pooled genome-wide CRISPR interference (CRISPRi) screen in Escherichia coli K-12 MG1655 carrying the P2 Old (Old) anti-phage defence system on plasmid pFD266. The EcoWG1 dCas9 sgRNA library was introduced and guide abundance quantified by amplicon sequencing at the input timepoint (T0) and after DAPG induction of dCas9 (T3), in two biological replicates each, alongside an sfGFP-expressing control (pFR66, in place of the defence system) and the conjugation-donor input library (E. coli MFDpir, pFR56). Read 1 (22 sequencing cycles, custom Read 1 primer) carries the 20 bp sgRNA guide and read 2 is a 12 bp unique molecular identifier (umi1+umi2). Comparing guide abundance identifies host genes whose knockdown modulates P2 Old activity.
Institut Pasteur