Method for constructing zebra fish asap1b gene-knockout mutant by using CRISPR/Cas9 technology

Abstract

The invention discloses a method for constructing a zebra fish asap1b gene-knockout mutant by using a CRISPR / Cas9 technology. The method specifically comprises the following steps: determining the position of an asap1b-gRNA-Cas9 target site where the asap1b gene is knocked out, carrying out in-vitro transcription to obtain asap1b-gRNA, introducing the asap1b-gRNA and Cas9mRNA into zebra fish fertilized eggs, and carrying out screening culture to obtain the stably-inherited asap1b gene-knockout mutant. According to the invention, according to an selected section of high-efficiency targeting sequence located at the first exon of an asap1b gene, the asap1b gene in the zebra fish is knocked out by using the CRISPR / Cas9 technology, and the asap1b gene-knockout zebra fish capable of being stablyinherited is generated. Three mutation types of asap1b gene-knockout zebra fish strains are obtained in total, Asap1b protein translation is terminated in advance in all the strains, and an animal model foundation is laid for researching functions of the asap1b gene.

Description

technical field [0001] The invention relates to the technical field of genetically engineered zebrafish mutants, in particular to a method for constructing zebrafish asap1b gene knockout mutants using CRISPR / Cas9 technology. Background technique [0002] The CRISPR / Cas system is an acquired immune defense system that exists in bacteria and archaea. When bacteria are infected by viruses, they will leave foreign gene fragments in their genomes as "memory antibodies" to identify re-invading viruses. . The CRISPR / Cas system consists of a CRISPR (Clustered regularly interspaced short palindromicrepeat) element and a gene encoding a series of Cas proteins, wherein the CRISPR element consists of multiple identical repeat sequences (repeats) and different spacers (spacers) alternately arranged. The working principle of the system is: bacteria rely on Cas protein to capture exogenous nucleic acid fragments and insert them into the spacer sequence of the genome CRISPR element, so tha...

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Problems solved by technology

However, there are few reports on transforming tuberculosis susceptibility genes in zebrafish models to study the pathophysiology of tuberculosis, and there is no report on the use of gene editing technology to generate zebrafish asap1b gene mutants. In addition, the number of asap1b transcripts is as high as 6. CRISPR / Cas9 technology makes modification of asap1b more difficult

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