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

摘要: Owing to its wide metabolic versatility and physiological robustness, together with amenability genetic manipulations high resistance stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range applications in both industrial environmental applications. However, applied synthetic biology engineering approaches are still limited by lack specific tools effectively efficiently regulate expression target genes. Here, we present single-plasmid CRISPR-interference (CRISPRi) system expressing nuclease-deficient cas9 gene under control inducible XylS/Pm system, along option adopting constitutively expressed guide RNAs (either sgRNA or crRNA tracrRNA). We showed that enables tunable, tightly controlled repression (up 90%) chromosomally genes encoding fluorescent proteins, either individually simultaneously. In addition, demonstrate this method allows suppressing essential pyrF ftsZ, resulting significantly low growth rates morphological changes respectively. This versatile expands capabilities current CRISPRi toolbox efficient, targeted controllable manipulation P. putida.