Cloning and functional verification of endogenous U6 promoters for developing an efficient CRISPR/Cas9-mediated...

The U6 promoter is a critical component of the CRISPR/Cas9 system, as it drives the transcription of single-guide RNAs (sgRNAs) to enable precise genome editing. Endogenous promoters typically exhibit higher transcriptional activity than their exogenous counterparts, which can significantly enhance editing efficiency. However, the endogenous U6 promoter in kenaf (Hibiscus cannabinus L.), an important fiber crop, has not yet been characterized.

Using the Arabidopsis U6-26 (AtU6-26) promoter as a reference, we performed a homologous sequence search and identified two candidate U6 promoters in kenaf, designated HcU6-1 and HcU6-14. Promoter fragments were amplified from the kenaf cultivar 'Fuhong 952' and cloned into a β-glucuronidase (GUS) reporter vector. Histochemical GUS staining assays revealed that both HcU6 promoters were transcriptionally active, with HcU6-14 showing significantly stronger expression levels compared to HcU6-1.

To further evaluate the utility of these promoters for genome editing, we constructed CRISPR/Cas9 vectors targeting the kenaf acetolactate synthase (ALS) gene, driven by either HcU6-14P or the exogenous cotton GbU6-9P promoter. Agrobacterium rhizogenes K599-mediated transformation was used to induce hairy roots, and mutation analysis of the ALS gene was performed via Sanger sequencing. Notably, targeted mutations in the ALS gene were detected in hairy roots transformed with the HcU6-14P-driven CRISPR/Cas9 vector, whereas no mutations were observed in roots transformed with the exogenous GbU6-9P promoter. These results demonstrate that the endogenous HcU6-14 promoter confers superior genome editing efficiency compared to the heterologous promoter, which facilitates the development of improved varieties with enhanced agronomic traits.