Abstract:
Objective The efficiency of CRISPR/Cas-mediated gene editing in plants is influenced by the transcriptional level of sgRNA. Endogenous promoters can enhance sgRNA transcription and improve gene editing efficiency. The U6 promoter is commonly used to drive sgRNA expression in CRISPR/Cas gene editing in dicottyledonous plants, however, studies on the U6 promoter in poplar remain scarce. This study aims to identify potent endogenous U6 promoters in poplar and optimize the CRISPR/Cas toolbox for this species.
Method Seven endogenous U6 promoters were identified and cloned from the poplar ‘84K’ (Populus alba × P. tremula var. glandulosa). Seven proPagU6::GUS fusion expression vectors were constructed and transformed into tobacco. The transcriptional activities of different PagU6 promoters were analyzed using histochemical staining and qRT-PCR. A CRISPR/Cas9 vector utilizing PagU6 to drive sgRNA was constructed and transformed into poplar.
Result Histochemical staining and qRT-PCR analyses revealed that all seven PagU6 promoters exhibited transcriptional activity, with PagU6-1 showing the highest activity. The gene editing vector, driven by the PagU6-1 promoter for sgRNA expression, successfully edited target genes in poplar, achieving a gene editing efficiency of 16.22%.
Conclusion This study identified PagU6-1 as an ideal endogenous promoter with high transcriptional activity for optimizing CRISPR/Cas gene editing tools, providing a critical technical foundation for enhancing gene editing efficiency in poplar.
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