A CRISPR/Cas9 system and its application for knocking out the DMRT1 gene at double gRNA sites in peltaceae

Abstract

The invention discloses a CRISPR / Cas9 system for knocking out the dmrt1 gene at double gRNA sites in yellow catfish. The CRISPR / Cas9 system includes the following steps: (1) Target site 1 is designed on the dmrt1 gene of yellow catfish On the first exon, the target site 2 is designed on the third exon; (2) design primers according to the target site sequence of step (1) to detect the accuracy of the target site in the broodstock, use dmrt1 E1 F and dmrt1 E1 R amplifies target site 1 and nearby sequences, and uses dmrt1 E3 F and dmrt1 E3 R to amplify target site 2 and nearby sequences; (3) Using pUC19‑gRNA‑scaffold plasmid as a template, use dmrt1 E1 gRNA F and gRNA R was used to amplify the gRNA1 fragment by PCR, and dmrt1 E3 gRNA F and gRNA R were used to amplify the gRNA2 fragment by PCR; using the above PCR product as a template, gRNA was obtained by in vitro transcription and purification; (4) the pXT7‑hCas9 linearized plasmid was used as Template, transcribed and synthesized Cas9 mRNA in vitro; (5) Microinjected Cas9 mRNA and two gRNAs into one-cell embryos of yellow catfish; (6) Detected the mutation type and calculated 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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