Large chromosomal segment deletions by CRISPR/LbCpf1‐mediated multiplex gene editing in soybean

The creation of new soybean varieties has been limited by genomic duplication and redundancy. Efficient multiplex gene editing and large chromosomal segment deletion through clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are promising strategies for overcoming these obstacles. CRISPR/Cpf1 is a robust tool for multiplex gene editing. However, large chromosomal excision mediated by CRISPR/Cpf1 has been reported in only a few non-plant species. Here, we report on CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting. The CRISPR/LbCpf1 system was optimized for direct repeat and guide RNA lengths in crispr RNA (crRNA) array. The editing efficiency was evaluated using LbCpf1 driven by the CaMV35S and soybean ubiquitin promoter. The optimized system exhibited editing efficiencies of up to 91.7%. Our results showed eight gene targets could be edited simultaneously in one step when a single eight-gRNA-target crRNA array was employed, with an efficiency of up to 17.1%. We successfully employed CRISPR/LbCpf1 to produce small fragments (<1 Kb) and large chromosomal segment deletions (10 Kb-1 Mb) involving four different gene clusters in soybean. Together, these data demonstrate the power of the CRISPR/LbCpf1 platform for multiplex gene editing and chromosomal segment deletion in soybean, supporting the use of this technology in both basic research and agricultural applications.