Disruption of <i>MIR396e</i> and <i>MIR396f</i> improves rice yield under nitrogen-deficient conditions

The microRNA miR396 directly represses GROWTH-REGULATING FACTORs (OsGRFs) and has been implicated in regulating rice yield and in nitrogen assimilation. Overexpressing the miR396 targets OsGRF4 and OsGRF6 improves rice yield via increased grain size and panicle branching, respectively. Here, we used CRISPR/Cas9 to assess the function of miR396 genes in rice. Knockout of MIR396ef (MIR396e and MIR396f), but not other isoforms, enhanced both grain size and panicle branching, resulting in increased grain yield. Importantly, under nitrogen-deficient conditions, mir396ef mutants showed an even higher relative increase in grain yield as well as elevated above-ground biomass. Furthermore, we identified OsGRF8 as a new target of miR396, in addition to the known targets OsGRF4 and OsGRF6. Disruption of the miR396-targeting site in OsGRF8 was sufficient to both enlarge grain size and elongate panicles. Our results suggest that rice-seed and panicle development are regulated by miR396ef-GRF4/6/8-GIF1/2/3 modules and that miR396ef are promising targets of genome editing for breeding environmentally friendly rice varieties that require less nitrogen fertilization.