CRISPR/Cas9 system for knocking out dmrt1 gene at double gRNA sites in yellow catfish and application

Abstract

The invention discloses a CRISPR / Cas9 system for knocking out a dmrt1 gene at double gRNA sites in yellow catfish. The CRISPR / Cas9 system comprises the following steps: (1) designing a target site 1 on a first exon of the dmrt1 gene of the yellow catfish, and designing a target site 2 on a third exon; (2) designing a primer according to a target site sequence in the step (1) to detect the accuracyof the target sites in parent fishes, amplifying the target site 1 and nearby sequences by using dmrt1 E1 F and dmrt1 E1 R, and amplifying the target site 2 and nearby sequences by using dmrt1 E3 F and dmrt1 E3 R; (3) performing PCR amplification on a gRNA1 fragment by using dmrt1 E1 gRNA F and gRNA R by taking pUC19-gRNA-scaffold plasmid as a template, performing PCR amplification on a gRNA2 fragment by using dmrt1 E3 gRNA F and gRNA R, and performing in-vitro transcription and purification by taking the PCR product as a template to obtain gRNA; (4) performing in-vitro transcription synthesis on Cas9 mRNA by taking pXT7-hCas9 linearized plasmid as a template; (5) performing microinjection on the Cas9 mRNA and the two gRNAs into a cell-stage embryo of the yellow catfish; and (6) detectingthe mutation type, and calculating the gene editing rate.

Description

technical field [0001] The invention relates to the fields of biotechnology and genetic breeding, in particular to a CRISPR / Cas9 system for knocking out the dmrt1 gene at double gRNA sites in yellow catfish and its application. 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 a...

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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