CRISPRi knockdown of essential genes in Pseudomonas aeruginosa

To understand molecular pathways responding to loss of essential proteins in the inner- or outer-membrane, we knocked down essential genes in the inner- and outer membrane using a CRISPRi approach in Pseudomonas aeruginosa. CRISPRi is an inducible system with which even essential genes can be targeted by a specifig sg-RNA. In this study, we investigated the transcriptomic changes upon loss of BamA, LptD and FtsH. Loss of BamA and LptD, both outer membrane proteins, resulted in strong upregulation of genes involved in LipidA modification and upregulation of several two component systems. Depletion of FtsH resulted in strong upregulation in genes responsible for amino acid biosynthesis and regulatory genes. Surprisngly, knockdown of bamA and lptD resulted in (mild) upregulation of H1-T6SS genes. Overall design: RNAseq experiment on three independent biological replicates was performed on sg-bamA, sg-lptD, sg-ftsH and sg-CTRL expressing P.aeruginosa dCas9 strains. RNA was isolated in the exponential growth phase.