Abstract:The problem of cadmium pollution in rice grain is a serious threat to human health. Disrupting the function of the cadmium transport gene OsNramp5 in rice can effectively reduce the accumulation of Cd in rice. To rapidly create low Cd-accumulating rice germplasm. In this study, we used CRISPR/Cas9 gene editing technology to knock out the cadmium transport gene OsNramp5 under the background of ChuanHui491 (R491), an indica restorer line of three-line hybrid rice with high-quality and disease-resistant. Various edited plants with different mutations were obtained, and two homozygous mutant lines (KO1 and KO2) without transgenic elements with single-target mutations were screened. Compared with wild-type R491, the cadmium content of brown rice in knockout lines KO1 and KO2 was significantly reduced by about 90% when planted in cadmium-polluted soil fields. The results of the agronomic trait investigation showed that there was no significant difference in the KO1 plants compared with wild-type R491, however, the KO2 plants had significant decreases in plant height, seed setting rate, and 1000-grain weight. Therefore, knockout of the cadmium transporter gene OsNramp5 by CRISPR/Cas9 gene editing technology can rapidly create new rice germplasm with low cadmium accumulation, and the new germplasm created in this study provides a new genetic resource for accelerating the breeding of safe rice varieties that can be planted in the cadmium-polluted field.