Enhancing heritable genome editing in soybean by optimizing promoter combinations for the LbCas12a system.

The CRISPR-Cas system, adapted from prokaryotic immune mechanisms, enables programmable DNA targeting and editing. Recent advancements include base editors and prime editors, expanding genetic research applications. The LbCas12a (Cpf1) system offers unique advantages, including simplified operation and multiplex editing, yet achieving stable heritable edits in soybean remains challenging due to its complex paleo-tetraploid genome and transformation constraints. Here we optimized promoter combinations for the LbCas12a editing system to maximize both editing efficiency in the initial (E0) generation and the frequency of homozygous or biallelic mutants in the subsequent (E1) generation. We identified prAtHSP70-1 (At5G02500), prAtEF1αA4 (At5G60390), and prGmUbi1 (Glyma.10G251900) as an effective promoter set, and demonstrated that LbCas12a–crRNA accumulation needs to exceed a defined threshold to achieve efficient target cleavage. These findings provide a practical strategy for enhancing heritable genome editing in soybean and may be broadly applicable to other crops.