A Robust CRISPR Interference Gene Repression System in Pseudomonas

Abstract

spp. are widely used model organisms in different areas of research. Despite the relevance of 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 spp. using a nuclease-null 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 and 300-fold repression in pyoverdine production in This inducible system enables the study of essential genes, as shown by depletions in ,, and that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first characterization of protospacer adjacent motif (PAM) site preferences of 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 dCas9, 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. Although 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, , and using the 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, accelerating the study and engineering of this widely used model organism.

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

“…This gene encodes the FtsZ protein that plays a key role in septum formation during bacterial cell division (Lutkenhaus and Addinall, ; Stricker and Erickson, ). Efficient repression of ftsZ leads to a filamentous cell phenotype, as previously described by Elhadi et al () and Tan et al (). In this case, we showed that, contrary to other reported CRISPRi systems, the very low leakiness of the CRISPRi modules constructed in this study does not result in any visible repression effect in the absence of the inducer.…”

“…A linear response of downregulation levels of a chromosomally expressed msf·gfp was observed as a function of the inducer (3‐ m Bz) concentration. This feature represents a substantial improvement to previously CRISPRi‐based approaches, which did not allow for titratable gene downregulation (Kim et al , ), sometimes exhibiting high leakiness (> 50%) in the absence of the corresponding inducers (Tan et al , ). Importantly, we also demonstrated the ability to simultaneously downregulate three genes in P. putida with this system.…”

“…DNA strand specificity is relevant for CRISPRi, and targeting the non‐template strand is crucial for efficient repression – whereas targeting the template DNA strand of the coding sequence is less effective or not effective at all (Larson et al , ). It has also been shown that the downregulation effect is the most efficient when dCas9 binds to the promoter sequence (Qi et al , ; Tan et al , ). Bacterial promoters can be predicted using freely available in silico tools (such as online tool BPROM from Softberry.com).…”

“…putida KT2440. Tan et al (), for instance, used a two‐plasmid CRISPR interference (CRISPRi) system, based on the type II dCas9 homologue of Streptococcus pasteurianus . In this example, dCas9 from S. pasteurianus could be harnessed for efficient CRISPRi‐mediated downregulation of genes, requiring specific protospacer adjacent motif (PAM) sequences (5′‐NNGTGA‐3′ or 5′‐NNGCGA‐3′, where N represents any nucleotide).…”

“…Engineered catalytically inactive variants of the Cas9 protein (dead Cas9, dCas9) have been shown to act as a transcription repressor in Pseudomonas strains, including P. putida KT2440. Tan et al (2018), for instance, used a two-plasmid CRISPR interference (CRISPRi) system, based on the type II dCas9 homologue of Streptococcus pasteurianus. In this example, dCas9 from S. pasteurianus could be harnessed for efficient CRISPRi-mediated downregulation of genes, requiring specific protospacer adjacent motif (PAM) sequences (5 0 -NNGTGA-3 0 or 5 0 -NNGCGA-3 0 , where N represents any nucleotide).…”