ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microbial Physiology and Metabolism
Volume 16 - 2025 | doi: 10.3389/fmicb.2025.1604430
Genomic Engineering in Rhizobium etli: implementation and evaluation of systems based on dCas9
Provisionally accepted
- Center for Genomic Sciences, National Autonomous University of Mexico, Cuernavaca, Mexico
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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.
Keywords: CRISPR-Cas9, CRISPRi, gene editing, gene repression, Rhizobiales
Received: 01 Apr 2025; Accepted: 06 Jun 2025.
Copyright: © 2025 Bellahsen, Díaz-Méndez and Romero. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: David Romero, Center for Genomic Sciences, National Autonomous University of Mexico, Cuernavaca, Mexico
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