The CRISPR-Cas9 system has been a go-to tool for targeted mutagenesis in crops. To further test its range of applications, the team of Yuhei Kanazashi from Hokkaido University in Japan aimed to use the CRISPR-Cas9 system to perform simultaneous targeted mutagenesis of a duplicated locus, GmPPD1 and GmPPD2, orthologs of Arabidopsis PEAPOD (PPD) gene in soybean (Glycine max), using only a single guide RNA.

Most of the transformed plants had mutations for the targeted loci. Analysis of T₁ and T₂ generations indicates that the mutations induced in the T₀ generation can be transmitted to subsequent generations. Mutations induced in T₁ plants can also be detected in succeeding generations. This indicates that induction of mutations during T₁ generation increases the occurrence of mutations in germ cells, ensuring the transmission of mutations to succeeding generations.

Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T₂ seeds examined. Double mutants with mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds.

These data indicate that continuous induction of mutations in advanced generations of transgenic plants enable efficient simultaneous mutagenesis in duplicated loci in soybean.

For more information, read the article in Plant Cell Reports.