Scientists have built a new method for artificial sperm-mediated mice that produce point mutations efficiently

In a recent study, Li Jinsong Research Group of the Institute of Biomedical and Cells of the Chinese Academy of Sciences and Yuan Wen Of the Long March Hospital affiliated with the Second Military Medical University, combined CRISPR-Cas9 gene editing technology and semi-cloning technology, realized the efficient preparation of mice with point mutations. The research results were published online in international academic journals
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the establishment of models of point-mutated mice, especially those with disease-caused gene-point mutations, is essential for studying the function of genes and the pathogenic mechanisms of disease, but editing embryonic stem cells through traditional congenital recombination takes time and effort. In recent years, direct injection of fertilized eggs through CRISPR-Cas9 has been available in mice with carrying point mutations, but it is inefficient to produce mice with carrying point mutations and there is an individual genotype nesting phenomenon. Prior research by Li Jinsong's research team established a semi-cloning technique for mice to obtain healthy mice by injecting them into eggs, and further optimized the technology to obtain monopolithic stem cells (also known as "artificial sperm") that produced semi-cloned mice steadily and efficiently, providing a new method for rapid preparation of mutant mouse models with a point of carrying. The researchers first used single-stranded oligopoly nucleotides (ssODN, length 99 bases) with point mutations as repair templates to be transferred to monolithic cells with CRISPR-Cas9, and found that the efficiency of iso-recombinant to obtain cells carrying mutant site decreased dramatically as the distance between the destination mutation site and the Cas9 enzyme cutting site (i.e., the DNA double-stranded disconnect site) increased. When the ≥ is 10bp, the use of ssODN as a template is too inefficient to detect and to obtain mutant cells carrying these site. To this end, the project team constructed a double-stranded carrier with point mutation as a template (length 258bp-2.1kb), and found that the same source recombination efficiency can be significantly improved, and found that when the carrier length is 1-1.5kb, the same source recombination efficiency is the highest. The researchers then built a cell line of monolithic cells carrying point mutations and efficiently obtained mouse models carrying point mutations through egg injections. The researchers suggest that in the simulation of disease-caused site mutations, ssODN can be used as a template when the mutation site is <10bp away from the DNA double-stranded disconnect site, and a carrier as a template is more efficient when the mutation site is ≥10bp away from the DNA double-stranded disconnect site.
, Wei Leixin and Wang Xiukun are the co-first authors of this article. (Source: Huang Xin, China Science Daily)