Two efficient CRISPR/Cas9 systems for gene editing in soybean

Area of Science:

• Agricultural Science
• Molecular Biology
• Biotechnology

Background:

• CRISPR/Cas9 is a key tool for crop improvement, but efficiency varies in complex genomes like soybean.
• Optimizing CRISPR/Cas9 delivery and expression is crucial for enhancing gene editing outcomes in plants.

Purpose of the Study:

• To compare the efficiency of a single transcriptional unit (STU) versus a two-component transcriptional unit (TCTU) CRISPR/Cas9 system for soybean genome editing.
• To evaluate these systems for multiplex gene editing targeting GmIPK1 and GmIPK2 involved in phytic acid synthesis.

Main Methods:

• Utilized CRISPR/Cas9 technology with two distinct systems: STU (SpCas9 and sgRNA from one promoter) and TCTU (SpCas9 and sgRNA from separate promoters).
• Employed a multiplex system targeting two soybean genes (GmIPK1, GmIPK2) using the hairy root transformation method.

Main Results:

• Both STU and TCTU systems achieved gene-specific editing in soybean.
• The TCTU system demonstrated higher indel frequencies for GmIPK1 editing (1-749 bp deletions) compared to STU.
• While both systems showed major exclusions for GmIPK2, STU exhibited lower editing efficiency.

Conclusions:

• Both STU and TCTU CRISPR/Cas9 systems are effective for soybean gene editing.
• The TCTU system is more efficient overall, whereas the STU system offers a smaller CRISPR/Cas cassette size.