Bellahsen Oussama, Díaz-Méndez Rafael, Romero David
Programa de Ingenería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Front Microbiol. 2025 Jun 24;16:1604430. doi: 10.3389/fmicb.2025.1604430. eCollection 2025.
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.
CRISPR-Cas9是一种用于基因编辑和调控的强大工具，有助于基因功能分析。在本研究中，我们开发了一种强大的CRISPR干扰（CRISPRi）系统，以精确调节菜豆固氮共生菌中的基因表达。该系统基于两个兼容的质粒（pBBR1MCS2-dCas9和含有特定引导RNA的pRhigRNA）。根据所使用的引导RNA，将这两种质粒导入菜豆共生菌中会导致四个靶基因[DsRedexpress、（在操纵子上）和]受到显著抑制。通过在不同靶位点使用不同的引导RNA，我们实现了高达90%的基因抑制。重要的是，单独表达dCas9或与引导RNA共表达时，均未观察到对生长的显著二次效应或毒性。该系统可用于进一步研究菜豆共生菌中必需基因的功能，或者可与其他基因表达元件或基因编辑工具整合，如用于根瘤菌目高级基因组工程的碱基编辑器。
