A Robust CRISPR Interference Gene Repression System in Pseudomonas.

Area of Science:

• Microbiology and Molecular Biology
• Gene Regulation and Genome Engineering

Background:

• Pseudomonas species are vital model organisms across diverse research fields.
• Existing protein depletion tools for Pseudomonas are limited, hindering research.
• A need exists for efficient and versatile gene repression systems in Pseudomonas.

Purpose of the Study:

• To develop and validate a CRISPR interference (CRISPRi) system for gene repression in Pseudomonas species.
• To characterize the protospacer adjacent motif (PAM) site preferences of Streptococcus pasteurianus dCas9.
• To enable the study of essential genes through inducible protein depletion.

Main Methods:

• Utilized a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, dCas9) for CRISPR interference.
• Demonstrated gene repression by measuring β-galactosidase activity and pyoverdine production.
• Performed in vivo characterization of dCas9 PAM site preferences.

Main Results:

• Achieved robust and titratable gene depletion, with up to 100-fold repression in P. aeruginosa and 300-fold in P. putida.
• Successfully depleted the essential FtsZ protein in P. aeruginosa, P. putida, and P. fluorescens, observing consistent phenotypic changes.
• Identified NNGCGA as a functional PAM site for S. pasteurianus dCas9, expanding its targeting range, especially in GC-rich genomes.

Conclusions:

• The developed CRISPRi system provides a powerful, inducible, and titratable method for gene repression in Pseudomonas species.
• This system surpasses existing methods by avoiding genomic modifications and enabling simultaneous repression of multiple genes.
• The identification of a new PAM site significantly broadens the applicability of S. pasteurianus dCas9 for Pseudomonas research and engineering.