Genomic engineering in : 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 , 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 led to significant repression of four target genes [DsRedexpress, , (on the operon) and ] 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 , or it can be integrated with other gene expression elements or gene editing tools, such as base editors for advanced genome engineering in Rhizobiales.