CRISPR/Cas9-Mediated Improvement of Major Rice Variety TBR225 for Low Cadmium Accumulation

Cadmium (Cd) contamination in rice poses significant health risks to consumers.This study aimed to reduce Cd accumulation in the elite Vietnamese rice variety TBR225 (TBR225) by editing the Natural Resistance-Associated Macrophage Protein 5 (OsNRAMP5) gene using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPRassociated 9 (Cas9) technology.We successfully generated OsNRAMP5 knockout mutants through Agrobacterium-mediated transformation.Sequencing analysis revealed various mutation types, including deletions, insertions, and substitutions, all resulting in premature stop codons or frameshift mutations.Three homozygous, transgene-free mutant lines were selected for further analysis.These lines exhibited significantly reduced Cd accumulation in roots (78.4-84.5%),shoots (72.3-83.8%),and grains (50.5-66.0%)compared to wild-type plants when exposed to different Cd concentrations.Importantly, the OsNRAMP5 mutations did not adversely affect major agronomic traits, including growth time, plant height, tiller number, grain yield, or amylose content.Additionally, the accumulation of essential micronutrients such as Fe and Zn remained unaffected in the mutant lines.Our results demonstrate the potential of CRISPR/Cas9-mediated OsNRAMP5 editing as an effective strategy for developing low-Cd accumulating rice varieties without compromising agronomic performance or nutritional quality.This approach could significantly contribute to enhancing food safety in regions facing Cd contamination challenges.