Multiplex Editing of OsMads26, OsBsr-d1, OsELF3-2 and OsERF922 with CRISPR/Cas9 Confers Enhanced Resistance to Pathogens and Abiotic Stresses and Boosts Grain Yield in Rice (Oryza sativa)

Rice (<i>Oryza sativa</i>) is one of the world's major staple foods. However, stable rice production is constrained by various biotic and abiotic and stresses. Breeding and cultivation of rice varieties with resistance to multiple pathogens and environmental stresses is the most effective strategy to mitigate the adverse effect of pathogen attacks and abiotic stresses. Recently, researchers have focused on the exploitation of CRISPR/Cas9 technology to manipulate some negative defense-regulator genes to generate rice varieties with broad-spectrum resistance against rice pathogens. In this study, four negative regulator genes of rice blast, <i>OsMads26, OsBsr-1</i>, <i>OsELF3-2</i> and <i>OsERF922</i>, were selected as CRISPR/Cas9 targets. By simultaneously knocking out all four genes via CRISPR/Cas9 technology, we created three <i>mads26/bsr-1</i>/<i>elf3-2</i>/<i>erf922</i> quadruple knockout mutants. Our results demonstrated that all quadruple mutants exhibited much higher resistance not only to rice blast and bacterial blight but also to drought and salt stresses than the wildtype. Interestingly, grain yield of all three quadruple mutants was also drastically increased by 17.35% to 21.95%. Therefore, this study provides a novel strategy to rapidly improve rice varieties with broad-spectrum resistance to pathogens, elevated tolerance to abiotic stresses and enhanced yield potential.