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

Area of Science:

• Plant Biotechnology
• Genomics
• Molecular Biology

Background:

• Soybean variety development is hindered by genomic duplication and redundancy.
• Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems offer solutions for multiplex gene editing and chromosomal deletion.
• CRISPR/Cpf1 is effective for multiplex gene editing, but its use for large chromosomal excision in plants is limited.

Purpose of the Study:

• To report CRISPR/LbCpf1-induced large chromosomal segment deletions in soybean using multiplex gene targeting.
• To optimize the CRISPR/LbCpf1 system for direct repeat and guide RNA lengths in CRISPR RNA (crRNA) arrays.
• To evaluate editing efficiency using LbCpf1 driven by different promoters.

Main Methods:

• Optimization of the CRISPR/LbCpf1 system for soybean.
• Multiplex gene targeting using a single eight-guide RNA (gRNA)-target crRNA array.
• Evaluation of editing efficiency with LbCpf1 driven by CaMV35S and soybean ubiquitin promoters.
• Induction of small fragment (<1 Kb) and large chromosomal segment deletions (10 Kb-1 Mb) in soybean.

Main Results:

• Optimized CRISPR/LbCpf1 system achieved editing efficiencies up to 91.7%.
• Simultaneous editing of eight gene targets was achieved with 17.1% efficiency using an eight-gRNA-target crRNA array.
• Successful induction of deletions ranging from <1 Kb to 1 Mb in four different gene clusters.

Conclusions:

• The CRISPR/LbCpf1 platform is powerful for multiplex gene editing and chromosomal segment deletion in soybean.
• This technology supports advancements in basic soybean research.
• The findings facilitate the development of improved soybean varieties for agricultural applications.