Abstract:The problem of excessive cadmium in rice grains seriously threatens human health. Disrupting the function of the cadmium transport gene OsNramp5 in rice can effectively reduce cadmium accumulation in rice. To rapidly create new rice germplasm with low cadmium accumulation, this study used CRISPR/Cas9 gene editing technology to knock out the cadmium transport gene OsNramp5 in the high-quality disease-resistant restorer line Chuanhui491 (R491) of three-line hybrid rice. Various edited plants with different mutations were obtained, and two homozygous mutant lines (KO1 and KO2) with single-target mutations without transgenic elements were selected. Compared with wild-type R491, the cadmium content of brown rice in knockout lines KO1 and KO2 were significantly reduced by about 90% when planted in cadmium-polluted soil fields. Agronomic trait investigation revealed no significant difference between the KO1 plants and wild-type R491, whereas the plant height, seed setting rate, and 1000-grain weight of KO2 mutant lines were significantly reduced. Therefore, knocking out the cadmium transporter gene OsNramp5 by CRISPR/Cas9 gene editing technology can quickly create new rice germplasm with low cadmium accumulation. The new germplasm created in this study provides novel genetic resources for accelerating the breeding of safe rice varieties that can be planted in the cadmium-polluted field.