An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida
Summary 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 environm...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2020-03-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.13533 |
| _version_ | 1818501588923187200 |
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| author | Christos Batianis Ekaterina Kozaeva Stamatios G. Damalas María Martín‐Pascual Daniel C. Volke Pablo I. Nikel Vitor A.P. Martins dos Santos |
| author_facet | Christos Batianis Ekaterina Kozaeva Stamatios G. Damalas María Martín‐Pascual Daniel C. Volke Pablo I. Nikel Vitor A.P. Martins dos Santos |
| author_sort | Christos Batianis |
| collection | DOAJ |
| description | Summary 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. |
| first_indexed | 2024-12-10T20:58:21Z |
| format | Article |
| id | doaj.art-da3c42375a4a4a6994da20d0a03f4ac3 |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-12-10T20:58:21Z |
| publishDate | 2020-03-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-da3c42375a4a4a6994da20d0a03f4ac32022-12-22T01:33:54ZengWileyMicrobial Biotechnology1751-79152020-03-0113236838510.1111/1751-7915.13533An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putidaChristos Batianis0Ekaterina Kozaeva1Stamatios G. Damalas2María Martín‐Pascual3Daniel C. Volke4Pablo I. Nikel5Vitor A.P. Martins dos Santos6Laboratory of Systems and Synthetic Biology Wageningen & Research University 6708Wageningen The NetherlandsThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark 2800Kgs. Lyngby DenmarkLaboratory of Systems and Synthetic Biology Wageningen & Research University 6708Wageningen The NetherlandsLaboratory of Systems and Synthetic Biology Wageningen & Research University 6708Wageningen The NetherlandsThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark 2800Kgs. Lyngby DenmarkThe Novo Nordisk Foundation Center for Biosustainability Technical University of Denmark 2800Kgs. Lyngby DenmarkLaboratory of Systems and Synthetic Biology Wageningen & Research University 6708Wageningen The NetherlandsSummary 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.https://doi.org/10.1111/1751-7915.13533 |
| spellingShingle | Christos Batianis Ekaterina Kozaeva Stamatios G. Damalas María Martín‐Pascual Daniel C. Volke Pablo I. Nikel Vitor A.P. Martins dos Santos An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida Microbial Biotechnology |
| title | An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida |
| title_full | An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida |
| title_fullStr | An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida |
| title_full_unstemmed | An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida |
| title_short | An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida |
| title_sort | expanded crispri toolbox for tunable control of gene expression in pseudomonas putida |
| url | https://doi.org/10.1111/1751-7915.13533 |
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