动物实验 z-bo 2020-09-03 409

　　The Li Jinsong Research Group of the Chinese Academy of Sciences Institute of Biochemistry and Cell Research and the Yunwen Research Group of Changzheng Hospital of the Second Military Medical University combined CRISPR-Cas9 gene editing technology with semi-cloning technology to achieve efficient production of point mutant mice.

　　Establishing point mutation mouse models, especially those with point mutations in disease-causing genes, is essential for studying gene function and disease-causing mechanisms, but homologous recombination must be performed through traditional methods of editing embryonic stem cells A lot of labor is required. Recently, point mutant mice can be obtained by directly injecting CRISPR-Cas9 into fertilized eggs, but the production efficiency of point mutant mice is not high, and idiotypic mosaicism is observed. The previous work of Li Jinsong's research team was to establish mouse orphan androgen haploid embryonic stem cells (AG-haESCs), and obtained semi-clonal technology, and injected them into eggs to obtain healthy mice. By further optimizing the technology, we have achieved stable and efficient semi-clonal mouse production. Haploid stem cells (also known as "artificial sperm") provide a new way to quickly prepare mouse models with target mutations. The researchers first used single-stranded oligodeoxynucleotides (ssODN, 99 bases in length) with point mutations as a repair template, then used CRISPR-Cas9 to transfer them to haploid cells and then target them. As the distance between the mutation site and the Cas9 enzyme cleavage site (ie, the DNA double-strand break site) increases, the efficiency of homologous recombination to obtain cells with the mutation site is greatly reduced. If the distance is 10 bp or greater, using ssODN as a template is not efficient and cannot be detected, and mutant cells with these sites cannot be obtained. To this end, the project team found that a double-stranded vector containing point mutations was created as a template (258 bp-2.1 kb in length), which can significantly improve the efficiency of homologous recombination. At the same time, when the vector length was 1-1.5 kb, we found homologous recombination. The most effective. After that, the researchers established a haploid cell line with point mutations, and effectively obtained a mouse model with target point mutations by injecting eggs. In the process of simulating pathogenic point mutations, if the distance between the mutation site and the DNA double-strand break site is less than 10 bp, researchers can use ssODN as a template. For single-strand breaks ≥10 bp, using a vector as a template can improve efficiency.