Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9
CRISPR-Cas9 is a powerful tool for gene editing and regulation, facilitating the analysis of gene function. In this study, we developed a robust CRISPR interference (CRISPRi) system to precisely modulate gene expression in the bacterium Rhizobium etli, the nitrogen-fixing symbiont of the common bean...
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| Language: | English |
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Frontiers Media S.A.
2025-06-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1604430/full |
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| author | Oussama Bellahsen Rafael Díaz-Méndez David Romero |
| author_facet | Oussama Bellahsen Rafael Díaz-Méndez David Romero |
| author_sort | Oussama Bellahsen |
| collection | DOAJ |
| description | CRISPR-Cas9 is a powerful tool for gene editing and regulation, facilitating the analysis of gene function. In this study, we developed a robust CRISPR interference (CRISPRi) system to precisely modulate gene expression in the bacterium Rhizobium etli, the nitrogen-fixing symbiont of the common bean. The system is based on two compatible plasmids (pBBR1MCS2-dCas9 and pRhigRNA containing specific guide RNAs). Introduction of both plasmids in R. etli led to significant repression of four target genes [DsRedexpress, recA, thiC (on the thiCOSGE operon) and rdsA] depending on the guide RNA used. By employing different guide RNAs at various target sites, we obtained up to 90% gene repression. Importantly, neither significant secondary effects on growth nor toxicity were observed upon expression of dCas9, either alone or in co-expression with the guide RNAs. This system can be utilized for further investigations on the function of essential genes in R. etli, or it can be integrated with other gene expression elements or gene editing tools, such as base editors for advanced genome engineering in Rhizobiales. |
| format | Article |
| id | doaj-art-d9a7d0f690ae4e8ca96001b5bbe043de |
| institution | DOAJ |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-d9a7d0f690ae4e8ca96001b5bbe043de2025-08-20T03:23:42ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-06-011610.3389/fmicb.2025.16044301604430Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9Oussama BellahsenRafael Díaz-MéndezDavid RomeroCRISPR-Cas9 is a powerful tool for gene editing and regulation, facilitating the analysis of gene function. In this study, we developed a robust CRISPR interference (CRISPRi) system to precisely modulate gene expression in the bacterium Rhizobium etli, the nitrogen-fixing symbiont of the common bean. The system is based on two compatible plasmids (pBBR1MCS2-dCas9 and pRhigRNA containing specific guide RNAs). Introduction of both plasmids in R. etli led to significant repression of four target genes [DsRedexpress, recA, thiC (on the thiCOSGE operon) and rdsA] depending on the guide RNA used. By employing different guide RNAs at various target sites, we obtained up to 90% gene repression. Importantly, neither significant secondary effects on growth nor toxicity were observed upon expression of dCas9, either alone or in co-expression with the guide RNAs. This system can be utilized for further investigations on the function of essential genes in R. etli, or it can be integrated with other gene expression elements or gene editing tools, such as base editors for advanced genome engineering in Rhizobiales.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1604430/fullCRISPR-Cas9CRISPRigene editinggene repressionRhizobiales |
| spellingShingle | Oussama Bellahsen Rafael Díaz-Méndez David Romero Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 Frontiers in Microbiology CRISPR-Cas9 CRISPRi gene editing gene repression Rhizobiales |
| title | Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 |
| title_full | Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 |
| title_fullStr | Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 |
| title_full_unstemmed | Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 |
| title_short | Genomic engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9 |
| title_sort | genomic engineering in rhizobium etli implementation and evaluation of systems based on dcas9 |
| topic | CRISPR-Cas9 CRISPRi gene editing gene repression Rhizobiales |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1604430/full |
| work_keys_str_mv | AT oussamabellahsen genomicengineeringinrhizobiumetliimplementationandevaluationofsystemsbasedondcas9 AT rafaeldiazmendez genomicengineeringinrhizobiumetliimplementationandevaluationofsystemsbasedondcas9 AT davidromero genomicengineeringinrhizobiumetliimplementationandevaluationofsystemsbasedondcas9 |