CRISPR/Cas9 multiplex genome editing to enhance grain lysine concentration in a US rice cultivar

Rice ( Oryza sativa L.) is a well-known staple crop providing daily calories to more than half of the world’s population. Its nutritional value is reduced due to insignificant levels of essential amino acids. Of the 20 amino acids, lysine is one of the most limiting factors, and low levels can lead to severe malnutrition issues affecting 25-30% of children worldwide. To address this, we knocked out the first bifunctional gene in the lysine catabolic pathway using the endogenous tRNA-processing CRISPR-Cas9 system, a method that has not been reported for increasing lysine levels in rice grains. A vector construct containing sgRNAs for the target genes was transformed into the U.S. elite rice variety Presidio using Agrobacterium -mediated transformation. A total of 19 transgene-positive T 0 plants with knockout mutations were confirmed. Four T 0 plants were advanced to the next generation based on transgene copy number, mutation pattern, and agronomic traits. Further screening confirmed a total of 7 transgene-free T 1 lines with homozygous edits. All lines showed a significant increase in lysine content compared to the wild-type cultivar Presidio. Progeny from three lines showed an almost 2-fold increase in lysine content without affecting morphological or agronomical traits. Furthermore, T 3 seeds showed a consistent increase in lysine content, confirming the heritability of the higher lysine trait across generations. The findings from our study establish a foundation for improving rice's nutritional value, thereby tackling two challenges: malnutrition and chronic disease prevention in both developing and developed countries. • Biofortified rice seeds with essential amino acids can create a balanced diet • CRISPR-Cas9 editing of the LKR/SDH genes can enhance lysine content in rice • T 2 seeds demonstrated nearly a 2-fold increase in lysine content versus wild-type • Enhancement of lysine in edited lines was stable across generations • This was achieved without impacting the agronomical traits of the rice plants