Abstract: Objective  To establish a leptin receptor (Lepr) and endothelial nitric oxide synthase (eNos) double-knockout (DKO) mouse model based on CRISPR/Cas9 gene editing technology, in order to construct a mouse model with advanced diabetic nephropathy. Methods The corresponding gRNA was prepared based on the eNos gene, and the CRISPR-Cas9 system was microinjected into the fertilized eggs of C57BL/Ks (BKS) background mice. The fertilized eggs were transferred into the fallopian tubes of female mice with pseudopregnancy. After birth, the F0 generation positive mice with the eNos^+/－ genotype were sorted out through polymerase chain reaction（PCR） identification and sequencing analysis, and Lepr gene heterozygous mice, Lepr－F0, under the BKS background were obtained. The eNos－F0 and Lepr－F0 generation mice were crossed to obtain eNos^+/－/Lepr^db/m double heterozygous F1 generation mice. The double heterozygous F1 generation mice were further mated to screen out Lepr and eNos double gene knockout mice. The mouse genotypes were identified using PCR method, and mice were divided into wild-type (WT) group and DKO group according to the gene identification results. The body weight, blood glucose, water and food intake of the mice in each group were monitored. The urinary albumin and urinary creatinine levels of the mice were detected with enzyme-linked immunosorbent assay（ELISA）, and the urinary albumin excretion rate was calculated. The pathological changes of the renal tissue of the mice in each group were examined with hematoxylin-eosin（HE） and periodic acid-silver metheramine（PASM） staining. Results PCR detection results showed that lepr^db/db/eNos^－/－ DKO mice were successfully constructed. Compared with the littermate control group, the body weight, blood glucose level, drinking water and food intake of DKO mice were significantly higher than those of the littermate control group. The urinary albumin and urinary albumin excretion rate of DKO mice were significantly higher than those of WT mice. The pathological results showed that the glomerular volume of DKO mice was significantly increased, and the mesangial matrix hyperplasia was obvious. Conclusions Lepr^db/db/eNos^－/－ DKO mice can be successfully constructed based on CRISPR/Cas9 gene editing technology. DKO mice can manifest the typical diabetic nephropathy and provide an animal model for in-depth study of the mechanisms of diabetic nephropathy.

Key words: gene knockout, CRISPR/Cas9 technology, diabetic kidney disease, eNos, Lepr