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 <i>Rhizobium etli</i>, 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 <i>R. etli</i> led to significant repression of four target genes [DsRedexpress, <i>recA</i>, <i>thiC</i> (on the <i>thiCOSGE</i> operon) and <i>rdsA</i>] 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 <i>R. etli</i>, or it can be integrated with other gene expression elements or gene editing tools, such as base editors for advanced genome engineering in Rhizobiales.