Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm

The efficient genome editing tool (the CRISPR/Cas12a system) has been used in research on plant functionional genomics and improvement of agronomic traits. In this study, CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. Using this system, crRNAs was driven by the Pol II pGhaGloA promoter to construct the pGhRBE3-pGhαGloA- GhPGF vector and carry out genetic transformation. The vector could work efficiently in all positive transgenic plants and the editing efficiency at the crRNA1 target site was up to 93.37%, and the editing efficiency of the crRNA2 target was up to 88.24%, which is significantly higher in editing efficiency of the pGhRBE3 system with Pol III promoter- Ubi 6.7 promoter, this result indicates that the Pol II promoter is more suitable for expressing multiple sgRNA or crRNA than the pol III promoter in cotton. The vector mainly generated the editing type of fragment deletion and the deletion size was in the range of 3-12 bp with the editing sites spanning at the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from T 0 to T 2 generation and three transgene-free lines with target site mutations of GhPGF gene were obtained and these glandless and gossypol-free/(low contents) cotton germplasm will play key role for healthy cottonseeds oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, which provides a powerful tool for cotton functionional genomics and genetic improvement.