A Robust CRISPR Interference Gene Repression System in Pseudomonas

Abstract

<i>Pseudomonas</i> spp. are widely used model organisms in different areas of research. Despite the relevance of <i>Pseudomonas</i> in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in <i>Pseudomonas</i> spp. using a nuclease-null <i>Streptococcus pasteurianus</i> Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in <i>P. aeruginosa</i> and 300-fold repression in pyoverdine production in <i>Pseudomonas putida</i> This inducible system enables the study of essential genes, as shown by <i>ftsZ</i> depletions in <i>P. aeruginosa</i>, <i>P. putida</i>, and <i>Pseudomonas fluorescens</i> that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first <i>in vivo</i> characterization of protospacer adjacent motif (PAM) site preferences of <i>S. pasteurianus</i> dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of <i>S. pasteurianus</i> dCas9, especially in GC-rich organisms.<b>IMPORTANCE</b><i>Pseudomonas</i> spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Although <i>Pseudomonas</i> spp. are widely used as model organisms in different areas of research, existing tools to deplete a protein of interest in these organisms remain limited. We have developed a robust and inducible gene repression tool in <i>P. aeruginosa</i>, <i>P. putida</i>, and <i>P. fluorescens</i> using the <i>Streptococcus pasteurianus</i> dCas9. This method of protein depletion is superior to existing methods, such as promoter replacements and addition of degradation tags, because it does not involve genomic modifications of the target protein, is titratable, and is capable of repressing multiple genes simultaneously. This gene repression system now enables easy depletion of specific proteins in <i>Pseudomonas</i>, accelerating the study and engineering of this widely used model organism.