Method for knocking out zebrafish p2rx2 gene

Abstract

The present invention relates to the technical field of gene knockout and particularly to a method for knocking out zebrafish p2rx2 gene. A CRISPR / Cas9 gene editing technology is used to design suitable target sites on the zebrafish p2rx2 gene and synthesizes specific sgRNA and Cas9-mRNA in vitro for zebrafish gene editing. The method can silence the specific gene more efficiently and accurately,is also simple to manufacture and low in cost, can also simultaneously cut multiple sites on the target gene and silences any number of single genes. The present invention also provides a method for gene knockout breeding of p2rx2 gene-deficient type zebrafish. The zebrafish model constructed by the method helps to further reveal entire processes of ear vesicle and hair cell morphogenesis and molecular mechanism regulating the processes. The method is of great significance for understanding pathogenesis of congenital deafness and research and development of a gene correction treatment scheme.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for knocking out the zebrafish p2rx2 gene. Background technique [0002] The p2rx2 gene is located on chromosome 5 of zebrafish, including 12 exons and 11 introns, the cDNA is 1292bp in length, encoding 400 amino acids, p2rx2 contains 4 evolutionarily conserved functional domains, and through genetic differences Expression profile analysis and genome association analysis found that p2rx2 was expressed in multiple tissues in early human embryos, especially in the ear vesicles. P2RX2 gene mutation can cause non-syndromic autosomal dominant deafness (DFNA41). P2RX2 gene is a candidate gene for noise susceptibility genes, and patients are highly sensitive to noise. [0003] The genes and signaling pathways in the development of zebrafish and human ear canal are highly homologous, and the p2rx2 gene is relatively conservative in evolution. Studies have found that the e...

AI Technical Summary

Problems solved by technology

[0005] However, the current general method for detecting CRISPR / Cas9 mutations in zebrafish gene knockout has certain defects. For example, it takes a lot of time and reagents to cut the tail of zebrafish in the F0 generation, and do TA cloning and Sanger sequencing. And the cost of sequencing, and the sequencing of the F0 generation may not necessarily be inherited to the next generation

Moreover, CRISPR / Cas9 technology requires many steps, the cost is too high, and the off-target rate is relatively high

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