CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida
Summary Implementation of single‐stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking co...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Wiley
2019-09-01
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| Series: | Microbial Biotechnology |
| Online Access: | https://doi.org/10.1111/1751-7915.13453 |
| _version_ | 1818775804713107456 |
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| author | Tomás Aparicio Víctor deLorenzo Esteban Martínez‐García |
| author_facet | Tomás Aparicio Víctor deLorenzo Esteban Martínez‐García |
| author_sort | Tomás Aparicio |
| collection | DOAJ |
| description | Summary Implementation of single‐stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking conspicuous phenotypes. Fortunately, the use of CRISPR/Cas9 as a device for counterselection of wild‐type sequences helps to overcome this limitation. Merging ssDNA recombineering with CRISPR/Cas9 thus enables a suite of genomic edits with a straightforward approach: a CRISPR plasmid provides the spacer DNA sequence that directs the Cas9 nuclease ribonucleoprotein complex to cleave the genome at the wild‐type sequences that have not undergone the change entered by the mutagenic ssDNA oligonucleotide(s). This protocol describes a complete workflow of the method optimized for P. putida, although it could in principle be applicable to many other pseudomonads. As an example, we show the deletion of the edd gene that encodes one key enzyme that operates the EDEMP cycle for glucose metabolism in P. putida EM42. By combining two incompatible CRISPR plasmids with different antibiotic selection markers, we show that the procedure can be cycled to implement consecutive deletions in the same strain, e.g. deletion of the pyrF gene following that of the edd mutant. This approach adds to the wealth of genetic technologies available for P. putida and strengthens its status as a chassis of choice for a suite of biotechnological applications. |
| first_indexed | 2024-12-18T11:02:52Z |
| format | Article |
| id | doaj.art-bc2fa6a5ddfb41dbbfc3573f7c9d0a6b |
| institution | Directory Open Access Journal |
| issn | 1751-7915 |
| language | English |
| last_indexed | 2024-12-18T11:02:52Z |
| publishDate | 2019-09-01 |
| publisher | Wiley |
| record_format | Article |
| series | Microbial Biotechnology |
| spelling | doaj.art-bc2fa6a5ddfb41dbbfc3573f7c9d0a6b2022-12-21T21:10:11ZengWileyMicrobial Biotechnology1751-79152019-09-011251076108910.1111/1751-7915.13453CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putidaTomás Aparicio0Víctor deLorenzo1Esteban Martínez‐García2Systems and Synthetic Biology Program Centro Nacional de Biotecnología (CNB‐CSIC) Campus de Cantoblanco 28049 Madrid SpainSystems and Synthetic Biology Program Centro Nacional de Biotecnología (CNB‐CSIC) Campus de Cantoblanco 28049 Madrid SpainSystems and Synthetic Biology Program Centro Nacional de Biotecnología (CNB‐CSIC) Campus de Cantoblanco 28049 Madrid SpainSummary Implementation of single‐stranded DNA (ssDNA) recombineering in Pseudomonas putida has widened the range of genetic manipulations applicable to this biotechnologically relevant bacterium. Yet, the relatively low efficiency of the technology hampers identification of mutated clones lacking conspicuous phenotypes. Fortunately, the use of CRISPR/Cas9 as a device for counterselection of wild‐type sequences helps to overcome this limitation. Merging ssDNA recombineering with CRISPR/Cas9 thus enables a suite of genomic edits with a straightforward approach: a CRISPR plasmid provides the spacer DNA sequence that directs the Cas9 nuclease ribonucleoprotein complex to cleave the genome at the wild‐type sequences that have not undergone the change entered by the mutagenic ssDNA oligonucleotide(s). This protocol describes a complete workflow of the method optimized for P. putida, although it could in principle be applicable to many other pseudomonads. As an example, we show the deletion of the edd gene that encodes one key enzyme that operates the EDEMP cycle for glucose metabolism in P. putida EM42. By combining two incompatible CRISPR plasmids with different antibiotic selection markers, we show that the procedure can be cycled to implement consecutive deletions in the same strain, e.g. deletion of the pyrF gene following that of the edd mutant. This approach adds to the wealth of genetic technologies available for P. putida and strengthens its status as a chassis of choice for a suite of biotechnological applications.https://doi.org/10.1111/1751-7915.13453 |
| spellingShingle | Tomás Aparicio Víctor deLorenzo Esteban Martínez‐García CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida Microbial Biotechnology |
| title | CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida |
| title_full | CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida |
| title_fullStr | CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida |
| title_full_unstemmed | CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida |
| title_short | CRISPR/Cas9‐enhanced ssDNA recombineering for Pseudomonas putida |
| title_sort | crispr cas9 enhanced ssdna recombineering for pseudomonas putida |
| url | https://doi.org/10.1111/1751-7915.13453 |
| work_keys_str_mv | AT tomasaparicio crisprcas9enhancedssdnarecombineeringforpseudomonasputida AT victordelorenzo crisprcas9enhancedssdnarecombineeringforpseudomonasputida AT estebanmartinezgarcia crisprcas9enhancedssdnarecombineeringforpseudomonasputida |