Method for mediating site-directed knock-in of goat Tbeta4 gene based on CRISPR/Cas 9 technology

Abstract

The invention employs a CRISPR-Cas9 system to complete mediation of a goat Tbeta4 gene for site-directed knock-in. A gRNA expression vector and a Tbeta 4 homologous recombinant vector based on the CRISPR-Cas9 system are constructed according to a CCR5 gene sequence of the goat. An optimized CRISPR-Cas9 vector and a constructed gRNA expression vector and the Tbeta 4 homologous recombinant vector are transferred to skeletal muscle satellite cells together, in order to obtain cells with the site-directed Tbeta4 gene. A targeting vector constructed based on the CRISPR-Cas9 system provides a simple, fast and safe pathway for site-directed knock-in of the goat Tbeta4 gene. Any screening marker genes are not related in a cell line screening process, safety of transgenic animals is greatly improved, and the method has important value for genetic breeding of goats and research of gene function.

Description

technical field

[0001] The invention relates to the fields of molecular biology and animal genetic breeding, in particular to a method for point-directed knock-in of goat Tβ4 gene mediated by CRISPER-Cas9 technology. Background technique

[0002] CRISPR-Cas9, as a site-specific gene modification technology, has developed rapidly in recent years. The technology is based on Clustered regulatory interspaced short palindromic repeat (CRISPR) and Cas9 nuclease (CRISPR associated system9, Cas9), which is the acquired immune system existing in bacteria and archaea, through its transcription The product crRNA (CRISPR RNA) and CRISPR-associated protein (CRISPR-associated, Cas), especially the Cas9 protein, can effectively help bacteria resist the infection of foreign DNA. Based on this principle, the CRISPR-Cas9 system has been transformed into a third-generation gene editing technology. This technology is guided by a short small RNA to modify DNA, cut the double strand of DNA to cr...

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