A Truncated Endogenous U6 Promoter Enables High-Efficiency CRISPR Editing in Flax ( 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 ( 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 was performed to measure the relative activity of dual luciferase. The 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 driving sgRNA. Agrobacterium-mediated infection of flax hypocotyls yielded transgenic albino flax shoots. DNA from these shoots was used as a template to amplify fragments, which were then sequenced. Sequencing analysis revealed that CRISPR/Cas9 vectors using achieved higher editing frequencies at compared to -driven systems.