The use of CRISPR/CAS9 as a reverse genetics tool to validate genome-wide association candidates

Genome‐wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used CRISPR/Cas9 nucleases together with randomized, well‐replicated experiments to evaluate the function of genes that had been identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. Eight candidate genes found in six clusters of strongly associated single nucleotide polymorphisms were evaluated and were found to have statistically significant effects on nodule production for two candidate genes. Annotated functions of these two genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid‐related defense response. These results demonstrate the utility of GWA combined with CRISPR/Cas9 mutagenesis to validate genes contributing to naturally occurring variation in quantitative traits.