Increasing the level of resistant starch in ‘Presidio’ rice through multiplex CRISPR–Cas9 gene editing of <i>starch branching enzyme</i> genes

Rice (Oryza sativa L.) is an excellent source of starch, which is composed of amylopectin and amylose. Resistant starch (RS) is a starch product that is not easily digestible and absorbed in the stomach or small intestine and instead is passed on directly to the large intestine. Cereals high in RS may be beneficial to improve human health and reduce the risk of diet-related chronic diseases. It has been reported through chemical mutagenesis and RNA interference studies that starch branching enzymes (SBEs) play a major role in contributing to higher levels of RS in cereal crops. In this study, we used multiplex clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated protein 9 (Cas9) genome editing to simultaneously target all four SBE genes in rice using the endogenous transfer RNA (tRNA)-processing system for expressing the single-guide RNAs (sgRNAs) targeting these genes. The CRISPR-Cas9 vector construct with four SBE gene sgRNAs was transformed into the U.S. rice cultivar Presidio using Agrobacterium-mediated transformation. Knockout mutations were identified at all four SBE genes across eight transgene-positive T0 plants. Transgene-free T1 lines with different combinations of disrupted SBE genes were identified, with several SBE-edited lines showing significantly increased RS content up to 15% higher than the wild-type (WT) cultivar Presidio. Although further efforts are needed to fix all of the mutant alleles as homozygous, our study demonstrated the potential of multiplex genome editing to develop high-RS lines.