Pseudomonas putida CRISPRi Gene Repression

CRISPR knockout uses Cas9 nuclease to create double-strand breaks that permanently disrupt the gene, while CRISPRi employs a catalytically dead dCas9 fused to a repressor domain to reversibly block transcription without altering the genome. CRISPRi is ideal for essential genes or when tunable control is needed, and it avoids selective pressure for loss-of-function mutations.

Yes, our system supports multiplex CRISPRi by co-expressing several sgRNAs from a single vector or using arrayed constructs. We can design and test up to five target genes per project. Repression efficiency for each target is validated individually by qRT-PCR.

Each strain undergoes sequence confirmation of the CRISPRi construct via Sanger sequencing, colony PCR to verify plasmid maintenance, and qRT-PCR to quantify target gene knockdown (typically project-dependent performance that is verified during development repression, depending on target and sgRNA). Growth curves are also recorded to evaluate any fitness effects.

Our system is primarily optimized for P. putida KT2440. Adaptation to other P. putida strains (e.g., P. putida M2, P. alloputida) is possible on a case-by-case basis and may require promoter or marker adjustments. We recommend discussing your specific strain during the initial consultation.