Gene editing is a new technology for site-specific modification of genome. Using this technology, we can locate a specific site in the genome, cut the target DNA and insert a new gene fragment at this site. The project not only simulates the natural mutation of genes, but also modifies and edits the original genome to truly complete gene editing. Next redbert (Beijing) Biotechnology Co., Ltd. will introduce the technology and principle of gene editing.

Traditional animal breeding methods are limited by provenance, require a lot of human, material and financial resources, and the breeding process is long. Moreover, the hybridization of different varieties is very difficult, and the breeding results are difficult to make a breakthrough.

1、 Principle of gene editing

The basic principle of modern genome editing technology is the same, that is, activating the natural repair mechanism of cells through specific DNA double strand breaks, including non homologous end connection and homologous recombination repair.

Non homologous end joining is a low fidelity repair process. During the repair and reconnection of broken DNA, random insertion or loss of bases will occur, resulting in frame shift mutation, that is, gene inactivation, so as to realize the knockout of target genes. If there is a foreign donor gene sequence, NHEJ mechanism connects it to the double strand break DSB site to achieve targeted knockout.

Homologous recombination repair is a relatively high fidelity repair process. In the presence of recombinant donors with homologous arms, through the homologous recombination process, the target gene spheres of donor members can be fully integrated into the target site without random insertion or loss. If DSB is generated on both sides of the gene, the original gene can be replaced in the presence of homologous donors.

2、 Genome editing technology

At present, there are three main gene editing technologies, namely, artificial nuclease mediated zinc finger nuclease (ZFN), transcription activating effector nuclease (talen) and RNA guided CRISPR CAS nuclease (crispr-cas9).

1. ZFN genome editing technology

ZFN technology is the first genome editing technology. Its function is based on the unique DNA sequence identification of zinc finger protein. In 1986, the DNA binding domain family cys2-his2 zinc finger module was first found in eukaryotic transcription factors. In 1996, Kim et al. Artificially linked zinc finger protein with nuclease for the first time. In 2005, urnov et al. Found that a pair of ZFNs connected by four zinc fingers could recognize a 24 BP specific sequence, revealing the application of ZFN in genome editing.

2. Talen genome editing technology

In 2009, researchers identified a transcription activator like effector in the plant pathogen Xanthomonas. The amino acid sequence of the protein nucleic acid binding domain has a relatively constant correspondence with the nucleic acid sequence of the target gene. The specific recognition of DNA sequence by tall was used to replace zinc finger protein in ZFN technology. It is more designable than ZFN, not affected by upstream and downstream sequences, and has a broader application prospect.

3. Crispr-cas9 gene editing technology

Talen has been successfully applied to specific gene mapping in yeast, mammals and plants. Compared with zinc finger nuclease system, it has great application advantages, but there are still some problems to be solved, such as off target effect and specific binding of talen to genome. These problems are related to chromosome localization and adjacent sequences.

3、 Advantages of gene editing

Compared with the traditional gene targeting technology based on homologous recombination and embryonic stem cell technology, the new gene editing technology retains the characteristics of targeted modification, can be applied to more species, and has the characteristics of high efficiency, short construction time and low cost.

4、 Lack of gene editing

Gene editing is a new technology, but it also has the problems of complex construction and price. Its targeting limits its development in gene therapy and other fields.

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