CRISPR/Cas9-based editing of OsNF-YC4/GmNF-YC4 promoter yields high-protein crops

Genome editing is a new breeding technology widely touted for transgene-free crop improvement; however, to date, the majority of derived traits are created through gene knockout. We describe a novel approach using gene editing to upregulate gene expression by removing negative repressor binding motifs. Our previous work demonstrated that ectopic expression of NF-YC4 increases protein content of leaves and seeds at the expense of carbohydrates. We detected several conserved motifs predicted to be bound by repressors in the promoter of rice and soybean NF-YC4 genes. Using CRISPR/Cas9 to edit the promoters of rice and soybean NF-YC4 genes, we deleted promoter fragments harboring repressor binding motifs. Those deletions resulted in decreased repressor binding, increased NF-YC4 expression, increased protein and decreased carbohydrates. Gene-edited plants showed up to 48% higher leaf protein and 15% increased seed protein. Moreover, we exemplify a general approach for upregulating gene expression through targeted genomic deletions.