Processing of double-stranded RNA precursors is essential in regulating gene
expression in plant development and stress response. Small RNA processing
requires the activity of a diverse group of components. However, in most
plant species, there are insufficient mutant resources to study each
component. Hence, the team of Shaun J. Curtin from the University of
Minnesota generated legume mutants that represent mutations in each loci
involved in small RNA processing in soybean (Glycine max) and alfalfa
(Medicago truncatula) using CRISPR-Cas9 and TALENs platforms.

A CRISPR-Cas9 reagent was used to create a bi-allelic double mutant for the
two soybean paralogs for the Double-stranded RNA-binding2 gene (Gmdrb2ab
mutant). Meanwhile, a CRISPR-Cas9-generated alfalfa mutant without the Hua
enhancer1 (MtHen1) gene was also developed. All mutations were found to be
transmissible.

On the other hand, TALENs was used to generate a mutation within the soybean
Dicer-like2 gene. CRISPR-Cas9 mutagenesis of the soybean Dicer-like3 gene
and the GmHen1a gene was observed, but were not transmitted to the next
generation.

Finally, a set of mutant combinations was generated by combining the
previously reported mutants with the Gmdrb2ab double mutant.

This study demonstrates the use of genome engineering platforms to generate
a collection of useful mutant plant lines for future study of small RNA
processing in legume crops.

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