Targeting OsNIP3;1 via CRISPR/Cas9: A Strategy for Minimizing Arsenic Accumulation and Boosting Rice Resilience

Arsenic (As) contamination in rice poses a significant threat to human health due to its toxicity and widespread consumption. Identifying and manipulating key genes governing As accumulation in rice is crucial for reducing this threat. The large NIP gene family of aquaporins in rice presents a promising target due to functional redundancy, potentially allowing for gene manipulation without compromising plant growth. This study aimed to utilize genome editing to generate knock-out (KO) lines of genes of NIP family (OsLsi1, OsNIP3;1) and an anion transporter family (OsLsi2), in order to assess their impact on As accumulation and stress tolerance in rice. KO lines were created using CRISPR/Cas9 technology, and the As accumulation patterns, physiological performance, and grain yield were compared against wild-type (WT) under As-treated conditions. KO lines exhibited significantly reduced As accumulation in grain compared to WT. Notably, osnip3;1 KO line displayed reduced As in xylem sap (71-74%) and grain (32-46%) upon treatment. Additionally, these lines demonstrated improved silicon (23%) uptake, photosynthetic pigment concentrations (Chl a: 77%; Chl b: 79%, Total Chl: 79% & Carotenoid: 49%) overall physiological and agronomical performance under As stress compared to WT. This study successfully utilized genome editing for the first time to identify OsNIP3;1 as a potential target for manipulating As accumulation in rice without compromising grain yield or plant vigor. Environmental Implications Arsenic contaminated soil poses a serious threat to rice crops, environment and human health. Rice cultivation itself exacerbates this issue, as flooding paddy fields favours arsenic mobilization. Rice absorbs and transports arsenic through specific transporters and harms the plant by stunting growth and reducing yield. Arsenic accumulates in grains thereby enters the food chain and cause health issues like cancer. Understanding and manipulating these transporters is the key to develop rice varieties with low arsenic uptake, safeguarding both the environment and human health.