Cloning and Functional Verification of Endogenous Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor ().

Castor () seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous promoter with robust function. Here, we searched for small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six snRNA genes. The promoters of these snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor promoter sequences and outlines a method for verifying the function of promoters in plants using the particle delivery system.