CRISPR interference (CRISPRi) is a robust gene silencing technique that is ideal for targeting essential and conditionally essential (CE) genes. CRISPRi is especially valuable for investigating gene function in pathogens such as P. aeruginosa where essential and CE genes underlie clinically important phenotypes such as antibiotic susceptibility and virulence. To facilitate the use of CRISPRi in diverse bacteria-including P. aeruginosa-we developed a suite of modular, mobilizable, and integrating vectors we call, "Mobile-CRISPRi." We further optimized Mobile-CRISPRi for use in P. aeruginosa mouse models of acute lung infection by expressing the CRISPRi machinery at low levels constitutively, enabling partial knockdown of essential and CE genes without the need for an exogenous inducer. Here, we describe protocols for creating Mobile-CRISPRi knockdown strains and testing their phenotypes in a mouse pneumonia model of P. aeruginosa infection. In addition, we provide comprehensive guide RNA designs to target genes in common laboratory strains of P. aeruginosa and other Pseudomonas species.
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