Generation of a multiplex mutagenesis population via pooled <scp>CRISPR</scp>‐Cas9 in soya bean

<jats:title>Summary</jats:title><jats:p>The output of genetic mutant screenings in soya bean [<jats:italic>Glycine max</jats:italic> (L.) Merr.] has been limited by its paleopolypoid genome. <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas9 as a mutant screening tool. Here, we report a pooled <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sg<jats:styled-content style="fixed-case">RNA</jats:styled-content> assessment, pooled transformation, sg<jats:styled-content style="fixed-case">RNA</jats:styled-content> identification and gene editing verification. We constructed 70 <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sg<jats:styled-content style="fixed-case">RNA</jats:styled-content>s at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized <jats:italic>gmric1/gmric2</jats:italic> double mutants with increased nodule numbers and <jats:italic>gmrdn1‐1/1‐2/1‐3</jats:italic> triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops.</jats:p>