Dual-gRNA site amh gene knockout method in pelteobagrus fulvidraco and application

Abstract

The invention discloses a dual-gRNA site amh gene knockout method in pelteobagrus fulvidraco. The method comprises the following steps of (1) designing a target site 1 on the first exon of a pelteobagrus fulvidraco amh gene, and designing a target site 2 on the fourth exon; (2) according to a target site point sequence design primer in the step (1), detecting the accuracy of the target site in parent fish, amplifying the target site 1 and a near sequence by using amh E1 F and amh E1 R, and amplifying the target site 2 and the near sequence by using amh E4 F and amh E4 R; (3) using a pUC19-gRNA-scaffold plasmid as a template, performing PCR amplification on a gRNA1 fragment by using amh E1 gRNA F and gRNA R, and performing PCR amplification on a gRNA2 fragment by using amh E4 gRNA F and gRNA R, wherein the PCR products are used as the template, in vitro transcription is performed, and purification is performed, so that gRNA is obtained; (4) using a pXT7-hCas9 linearization plasmid as atemplate, and performing in vitro transcription to synthetize Cas9 mRNA; (5) performing microinjection on Cas9 mRNA and two gRNA into a cell stage embryo of pelteobagrus fulvidraco; and (6) detectingthe mutation type, and calculating the gene editing rate. The invention further discloses an application of the method.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic breeding, in particular to a method and application for knocking out the amh gene at double gRNA sites in yellow catfish. Background technique [0002] Pelteobagrus fulvidraco belongs to Osteichthyes, Siluriformes, Bagridae, and Pelteobagrus. It is one of the important freshwater economically farmed fishes in my country. Because the growth rate of male yellow catfish is 2-3 times that of female fish. In recent years, all-male yellow catfish farming has been promoted in China, but with the rapid development of the yellow catfish farming industry, some problems have emerged one after another. For example, the supply of YY super male fish used to produce all male fry is tight and expensive; in addition, due to inbreeding The phenomenon is serious, leading to the degradation of the germplasm of the yellow catfish, the reduction of growth rate, and the reduction of disease and stress resistan...

AI Technical Summary

Problems solved by technology

The traditional single-target knockout has low targeting efficiency, high uncertainty of DNA self-repair, easy to cause nonsense mutations, and the deletion of several bases is not easy to identify, often requiring a lot of money for sequencing identification

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