spp. are widely used model organisms in different areas of research. Despite the relevance ofin many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression inspp. using a nuclease-nullCas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity inand 300-fold repression in pyoverdine production inThis inducible system enables the study of essential genes, as shown bydepletions in,, andthat led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the firstcharacterization of protospacer adjacent motif (PAM) site preferences ofdCas9 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 ofdCas9, especially in GC-rich organisms.spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Althoughspp. 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,, andusing thedCas9. 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, accelerating the study and engineering of this widely used model organism. Pseudomonas Pseudomonas Pseudomonas Streptococcus pasteurianus P. aeruginosa Pseudomonas putida ftsZ P. aeruginosa P. putida Pseudomonas fluorescens in vivo S. pasteurianus S. pasteurianus Pseudomonas Pseudomonas P. aeruginosa P. putida P. fluorescens Streptococcus pasteurianus Pseudomonas IMPORTANCE
