Method for knocking off myo7ab gene of zebra fish

A zebrafish and gene technology, applied in the field of gene knockout, can solve the problems of high cost, many steps, time-consuming, reagent and sequencing costs, etc.

Inactive Publication Date: 2020-05-15
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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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

Method used

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  • Method for knocking off myo7ab gene of zebra fish
  • Method for knocking off myo7ab gene of zebra fish
  • Method for knocking off myo7ab gene of zebra fish

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Embodiment 1

[0043] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0044] Query the genomic DNA sequence of the zebrafish myo7ab gene on the National Center for Biotechnology Information (NCBI), analyze its functional domain on the website SMART (http: / / smart.embl-heidelberg.de / ), and knock out it according to CRISPR / Cas According to the principle, the target site of the myo7ab gene was designed on the website The ZiFiT Targeter (http: / / zifit.partners.org / ZiFiT / ). The selection of targets must follow this standard: 5'-GG-(N)18-NGG-3'. The GG dinucleotide at the 5' end is part of the T7 promoter, and this restriction is not required when designing the target site, but it must be ensured that the 3' end of the target site is NGG. The selected position of the target must be within the structural domain of the gene to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the myo7ab gene, thereby changing the expr...

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Abstract

The invention relates to the technical field of gene knockoff and in particular relates to a method for knocking off the myo7ab gene of zebra fish. According to the method, an appropriate targeting site is designed on the myo7ab gene of zebra fish by using a CRISPR/Cas9 (clustered regularly interspaced shortpalindromic repeats-associated 9) gene edition technique, and specific sgRNA (small guide ribonucleic acid) and Cas-9mRNA (messenger ribonucleic acid) are synthesized in vitro for gene edition of the zebra fish. By adopting the method, specific genes can be efficiently and precisely silenced, in addition, the preparation is simple, the cost is low, multiple sites on the target gene can be sheared simultaneously, and any number of single genes can be silenced. The invention further provides a method for knocking off genes to breed myo7ab gene deleted zebra fish. The zebra fish model constructed by using the method is helpful in further revealing the whole process of generation of otic bulla and capillary cellular morphologies and molecule mechanisms for regulating and controlling the process, and has very great significances for understanding on medical congenital deafness nosogenesis and research on treatment schemes of gene correction.

Description

technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for knocking out the zebrafish myo7ab gene. Background technique [0002] The myo7ab gene is located on chromosome 21 of the zebrafish, including 46 exons and 47 introns. The full-length cDNA is 6614bp, encoding 2204 amino acids. Myo7ab contains 12 evolutionarily conserved functional domains. Expression profile analysis and genome association analysis found that myo7ab is expressed in multiple tissues in the early human embryo, especially in the ear canal. The zebrafish myo7ab gene is homologous to human myosin 7A (MYO7A). MYO7A protein is located in inner ear hair cells and their tip stereocilia, and can affect the development, location and function of stereocilia. MYO7A mutations can be caused by human Usher syndrome, and can also cause non-syndromic autosomal dominant deafness (DFNA11) and chromosomal recessive deafness (DFNB2). [0003] The genes and signalin...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/90C12N9/22A01K67/027
CPCA01K67/0275A01K2217/075A01K2227/40A01K2267/02C12N9/22C12N15/113C12N15/907C12N2310/10
Inventor 谢鼎华赖若沙杨曙谢华平曾婷谢缤灵付贵芳邓慧玲胡昱瑶
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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