CRISPR-Cas9技术,遗传疾病 z-bo 2020-12-14 332

　　Genetic diseases refer to diseases caused by changes in genetic material, which can be passed on to offspring through fertility and are an important type of disease that plagues human health. The method to radically cure genetic diseases is to repair the altered genetic material in the germ cells through gene therapy, and pass the correct genetic material to the next generation to produce healthy individuals, thereby completely eliminating genetic defects in the population. However, currently existing genetic modification methods cannot effectively perform genetic editing in germ cells. The internationally renowned academic journal "Cell Research" Cell Research published online a collaborative study between the Li Jinsong research group and Wu Ligang research group of the Institute of Biochemistry and Cell, Chinese Academy of Sciences Shanghai Academy of Biological Sciences, and the Tang Fuchou research group of Peking University. Researchers use CRISPR-Cas9 technology The genetic defects were repaired in the spermatogonial stem cells of the mice, resulting in completely healthy offspring. This research provides a new idea for human gene therapy. CRISPR-Cas9 technology uses a single guide RNA with the same target sequence to guide Cas9 nuclease to recognize and cut specific targeted DNA, causing DNA double-strand or single-strand breaks. Then, the cell will use the two types of DNA it has. DNA repair mechanism repairs broken DNA, namely non-homologous end joining (NHEJ) or homology-directed repair (Homology-directed repair, HDR). This technique is simple and efficient. Since its introduction, it has been widely used in various model organisms to establish genetic modification models. Li Jinsong's research group cured mice's cataract genetic disease by directly injecting the CRISPR-Cas9 system into fertilized egg embryos carrying genetic defects (Wu et al; Cell Stem Cell, 2013, 13:659), and demonstrated for the first time that CRISPR-Cas9 technology can Efficiently used in the treatment of genetic diseases. However, this method of direct embryo injection has two problems: one is that the probability of newborn mice being cured is low, about 30%; the other is that there are a small number of experimental off-target phenomena.

　　 On this basis, in order to better solve these problems, the researchers obtained spermatogonial stem cells carrying pure and genetic mutations from the testes of cataract mice. Spermatogonial stem cells are a type of germline stem cells that can be cultured in vitro for a long time, can maintain stable epigenetic characteristics, and can produce functional gametes in recipient mice. The researchers transferred CRISPR-Cas9 into the spermatogonial stem cell line, and established a series of cell lines derived from a single spermatogonial stem cell by means of single cell expansion. Subsequently, the researchers conducted an in-depth analysis of these cell lines and selected cell lines that meet the following three conditions for transplantation experiments: 1) through genotype analysis to determine that both mutation sites have been repaired; 2) through predictive off-target site sequencing Or whole genome sequencing confirms that there is no off-target problem; 3) Through specific imprinted gene methylation identification or whole genome methylation sequencing, it is determined that the repaired spermatogonial stem cells maintain normal epigenetic characteristics. Finally, after transplanting these "high-quality" cells into the testis of recipient mice from which germ cells were removed, the researchers obtained 100% fully healthy mice.

　　 CRISPR-Cas9 technology-mediated genetic repair of germ cells provides a new idea for human gene therapy. Future research needs to verify the feasibility of this technical route in human germ cells.