A Truncated Endogenous U6 Promoter Enables High-Efficiency CRISPR Editing in Flax (Linum usitatissimum L.).

Functional U6 promoters are widely utilized in CRISPR gene editing systems for crops. The identification of endogenous U6 promoter activity and the establishment of CRISPR/Cas9 gene editing systems in various crops can enhance the efficiency and accuracy of gene editing in molecular breeding. In this study, four U6 snRNAs were identified in the genome of the oil flax (Linum usitatissimum L.) cultivar Longya 10, which exhibit high homology with the promoter regions of Arabidopsis thaliana U6 snRNA. We cloned and constructed fusion expression vectors with U6 promoter-driven dual-luciferase reporter genes. Transient transformation of flax and Nicotiana benthamiana was performed to measure the relative activity of dual luciferase. The U6-4 on chromosome 14 showed the highest transcriptional activity. Truncations of varying lengths from the 5' end of this promoter were tested, revealing that a 342 bp U6 promoter fragment possesses high transcriptional activity and an optimal length. Subsequently, we constructed a CRISPR/Cas9 gene editing vector with LuU6-5P/AtU6-P driving LusPDS sgRNA. Agrobacterium-mediated infection of flax hypocotyls yielded transgenic albino flax shoots. DNA from these shoots was used as a template to amplify LusPDS fragments, which were then sequenced. Sequencing analysis revealed that CRISPR/Cas9 vectors using Lu14U6-4-5P achieved higher editing frequencies at LusPDS compared to AtU6-P-driven systems.