Abstract: Objective　We generated ob mice (C57BL/6N-Lepem1 /Nifdc) with Lep gene knockout (ob/ob) using the CRISPR/Cas9 system, to establish a suitable animal model for preclinical drug evaluation for clinical diseases such as diabetes. Methods　According to the principle of CRISPR/Cas9 target design, single guide RNA targeting the mouse Lep gene was designed for transcription in vitro, and microinjected with Cas9 mRNA into mouse zygotes. Mouse tail DNA was extracted and detected by polymerase chain reaction and sequencing, followed by mating of positive and wild-type mice. Blood biochemistry and liver pathology were assessed in homozygous ob mice. Results　Eight positive mice were identified and a stable mouse strain was selected for further breeding. Serum triglycerides, total cholesterol, and alanine aminotransferase levels were significantly higher in homozygous ob mice than in wild-type mice, and liver pathology showed inflammatory infiltration and lipid vacuolar transformations. Conclusions　We successfully established a Lep gene knockout mouse model, which will provide an important addition to the national rodent experimental animal database and an animal model for preclinical drug evaluation.

Abstract:Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank. sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C57BL/6 mice. After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile, real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice. Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas9. After microinjection, miRNA-29b1 gene-mutated mice were obtained. The sequencing results showed that there were two types of genotype for the mutated mice, one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion. Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly. Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR/Cas9 technology.

Abstract:Objective To study the relationship of insulin receptor substrate-1 (Irs1) and metabolic disease, we generated Irs1 gene knockout rat by CRISPR/Cas9 system. Methods Two sgRNA targeting sites were designed for Irs1 targeting. The Cas9 and sgRNAs were transcribed by T7 RNA polymerase in vitro. Cas9 mRNA and sgRNA mixtures were pooled and microinjected into one-cell fertilized eggs of SD rats to generate rats with targeted mutation. Results Five rats with the mutations were detected with the efficiency of 83%. Conclusion The Irs1 gene knockout rats generated in this study can be transmitted by germline.

Abstract:Animal models are highly valuable systems that have been extensively used to elucidate human disease pathogenesis and to find therapeutic ways to treat human diseases. Since non-human primates are close to humans,monkeys are important model species in exploring the mechanisms and treatment of human neurodegenerative diseases, neuropsychiatric disorders, cognitive function, and neural circuits. However, due to the lack of embryonic stem cell lines in large animals, the traditional gene targeting technology is difficult to establish primate animal models of human diseases. CRISPR/Cas9, as a recently developed tool for genome modifications, has been successfully used to target genomic loci in mouse, rat, monkey, and other species. Here, we discuss the utilization of CRISPR/Cas9 technology in establishing monkey models for studying human neurodegenerative diseases.