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

C. Batianis, Ekaterina Kozaeva, S. Damalas, Maria Martín-Pascual, Daniel Volke, Pablo I. Nikel*, V.A.P. Martins dos Santos*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

19 Citations (Scopus)


Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.
Original languageEnglish
Pages (from-to)368-385
JournalMicrobial Biotechnology
Issue number2
Early online date11 Feb 2020
Publication statusPublished - Mar 2020


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