Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi

Michelle A Yu; Amy B Banta; Ryan D Ward; Neha K Prasad; Michael S Kwon; Oren S Rosenberg; Jason M Peters

doi:10.1007/978-1-0716-3473-8_2

Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi

Methods Mol Biol. 2024:2721:13-32. doi: 10.1007/978-1-0716-3473-8_2.

Authors

Michelle A Yu¹, Amy B Banta^{2

3}, Ryan D Ward^{2

4}, Neha K Prasad^{5

6}, Michael S Kwon¹, Oren S Rosenberg^{5

6

7}, Jason M Peters^{8

9

10

11}

Affiliations

¹ Division of Pulmonary, Critical Care, Allergy, and Sleep Medicine, Department of Medicine, University of California, San Francisco, CA, USA.
² Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA.
³ Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA.
⁴ Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA.
⁵ Chan Zuckerberg Biohub, San Francisco, CA, USA.
⁶ Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.
⁷ Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA.
⁸ Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA. jason.peters@wisc.edu.
⁹ Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA. jason.peters@wisc.edu.
¹⁰ Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA. jason.peters@wisc.edu.
¹¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA. jason.peters@wisc.edu.

PMID: 37819512
DOI: 10.1007/978-1-0716-3473-8_2

Abstract

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.

Keywords: CRISPR-Cas9; CRISPRi; Essential genes; Murine pneumonia model; Pseudomonas putida; Pseudomonas syringae; Systems biology.

MeSH terms

Animals
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Silencing
Mice
Phenotype
Pseudomonas aeruginosa* / genetics