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

A yellow catfish and gene technology, applied in the fields of biotechnology and genetic breeding, can solve the problems of high cost, nonsense mutation, and high uncertainty of DNA self-repair, and achieve the effect of reducing identification cost and improving knockout efficiency

Active Publication Date: 2020-01-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

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Embodiment 1

[0036] Step (1) Target site design:

[0037] The genomic DNA sequence and its mRNA sequence of the amh gene of the yellow catfish were queried on NCBI. The full length of the amh gene of the yellow catfish was 4660bp, including 7 exons and 6 introns. Design a target site for each of the first exon and the fourth exon, the sequence of the target site is shown in Table 1, and the structure of the target site is shown in figure 1 shown. The target sequence was compared by Blast on the NCBI website to verify the specificity of the target site.

[0038] Target site selection principles:

[0039] A. The target site contains 20 bases, of which the 5' end should be GG. This is because the gRNA used in the present invention is transcribed in vitro using a T7 promoter, and the T7 promoter requires the first two digits of the transcription start site to be GG , and the third place is preferably G or A.

[0040] B. The 3 bases immediately adjacent to the 3' end of the target site cons...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12Q1/6858A01K67/027
CPCC12N15/113C12N15/902C07K14/461C12Q1/6858A01K67/0276C12N2310/20A01K2207/15A01K2217/075A01K2227/40A01K2267/02C12Q2531/113C12Q2535/101C12Q2565/125
Inventor 卢建国李石竹方文宇
Owner SUN YAT SEN UNIV
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