Efficiency of CRISPR/Cas9 gene editing at multiple sites in soybean using one guide RNA

Mutation frequencies of 64.71% for GmPDS, 60.0% for GmFAD2, and 42.86% for GmALS were achieved using a single sgRNA CRISPR/Cas9 system in soybean.

• The CRISPR/Cas9 system was optimized to assess mutation efficiency in soybean's multiple-copy genes.
• Simultaneous site-directed mutagenesis was successfully performed at two homoeologous loci with one sgRNA.
• Chimeric and heterozygous mutations were the most common outcomes observed.
• Phenotypic associations were identified with mutation patterns at specific target loci.
• The findings indicate that while effective, the system exhibits relatively low transformation efficiency.

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