Lettuce (Lactuca sativa L.) is an important leafy vegetable crop, yet the efficiency and reliability of genome editing platforms in lettuce remain limited, particularly for precision base editing applications. In this study, we established an optimized PEG-mediated protoplast transformation system for lettuce through systematic evaluation of key parameters, including protoplast density, incubation time, plasmid size, and transformation method. Under optimized conditions, a maximum transient transformation efficiency of up to 81% was achieved. Using this optimized protoplast platform, we comparatively evaluated the performance of three single-base editing systems—adenosine base editor (ABE), glycosylase-based guanine base editor (gGBE), and rice alkylpurine DNA glycosylase-mediated A-to-K base editor (rAKBE)—targeting the LsALS gene, encoding acetolactate synthetase as a herbicide target with great value in weed control. Among the tested editors, ABE exhibited the highest A-to-G editing efficiency, reaching 9.3%. In contrast, gGBE and rAKBE showed lower editing efficiencies. Together, this study established a robust and reproducible protoplast-based platform for transient genome editing in lettuce and provides a practical framework for the rapid evaluation of base editing tools and target sites, firstly for gGBE and rAKBE evaluation in lettuce. The optimized system facilitates functional genomics studies and supports the development of precision breeding strategies in lettuce.
Jia et al. (Thu,) studied this question.