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.

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