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CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system-mediated goat EDAR (ectodysplasin-areceptor) gene knockout method

A gene knockout and goat technology, applied in the fields of molecular biology and animal genetics and breeding, can solve problems such as unreported research and achieve the effect of improving safety

Inactive Publication Date: 2017-07-25
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] No report on knockout of goat EDAR gene using CRISPER-Cas9 system

Method used

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  • CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system-mediated goat EDAR (ectodysplasin-areceptor) gene knockout method
  • CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system-mediated goat EDAR (ectodysplasin-areceptor) gene knockout method
  • CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system-mediated goat EDAR (ectodysplasin-areceptor) gene knockout method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Optimization of CRISPER-Cas9 vector.

[0034] Optimize the CRISPR-Cas9 expression vector purchased from Beijing Zhongyuan Company. The nucleotide sequence of the optimized CRISPER-Cas9 vector (i.e. hCas9 plasmid) is shown in SEQ ID NO: 1. For the hCas9 plasmid map, see figure 1 .

Embodiment 2

[0035] Example 2 Construction of gRNA expression vector.

[0036] According to the goat EDAR gene sequence (Gene ID: 102179206), the sgRNA sequence was designed for exon 6 of EDAR, and the gRNA expression vector based on the CRISPER-Cas9 system was constructed. The gRNA expression vector includes four parts: U6 promoter, target sequence, gRNA backbone and termination signal. The pattern diagram of goat EDAR gene knockout based on CRISPR-Cas9 system is shown in figure 2 . Wherein, the DNA sequence of the sgRNA action site is as follows: sgRNA1: 5'-GGAGAACTTCTCCGCGGGGC-3', sgRNA2: 5'-CGGCGCCACAAGGACTGCGA-3'. Use biological software to design sgRNA sequences according to the sgRNA action sites (sgRNA1 target site and sgRNA2 target site), clone them into gRNA expression vectors, transform Escherichia coli Trans-110, pick a single colony after plating, and carry out bacterial liquid PCR, identified by electrophoresis and sequencing, the single colony with correct sequencing was...

Embodiment 3

[0037] Example 3 Transfection of goat fetal fibroblasts by electroporation.

[0038] Thawed goat fetal fibroblasts were inoculated into 100mm culture dishes, and cultured in DMEM / F12 medium containing 10% fetal bovine serum in an incubator at 37°C, 5% CO2, and 100% relative humidity. When the cells reach 90% confluence, wash the cells with PBS, then digest them with 0.25% trypsin for 2-3 minutes, then stop the digestion with DMEM / F12 medium containing 10% fetal bovine serum, and blow gently Adherent cells were separated from the wall of the culture dish to form a cell suspension and collected in a 10mL centrifuge tube, centrifuged at 1500rpm for 5 minutes. After the centrifugation, remove the supernatant culture solution, add PBS to resuspend the cells, and then centrifuge at 1500rpm for 5 minutes to wash the cell pellet twice. Finally, after washing the cell pellet with Opti-MEM culture medium, resuspend the cells with an appropriate amount of Opti-MEM culture medium to reac...

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Abstract

The invention relates to a method to knock out goat EDAR (ectodysplasin-areceptor) gene via CRISPER-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR associated protein 9) system mediation; the method comprises: constructing two gRNA expression vectors based on CRISPER-Cas9 according to goat's EDAR gene sequences, transferring optimized CRISPER-Cas9 vectors and the constructed gRNA expression vectors jointly into fibroblasts of a goat fetus to obtain goat cells with EDAR gene knocked out. The CRISPER-Cas9-mediated targeting vectors constructed herein provide a simple, quick and safe means to knock out goat EDAR gene. The method involves no selection marker genes during cell line screening, the safety of transgenic animals is improved greatly, and the method is of great value to the genetic breeding of goat and genetic function studies.

Description

technical field [0001] The invention relates to the fields of molecular biology and animal genetic breeding, in particular to a method for knocking out the goat EDAR gene mediated by a CRISPER-Cas9 system. Background technique [0002] Modern gene editing technologies include: zinc finger nuclease (ZFNs), transcription activator-like effector nuclease (TALENs) and CRISPR-Cas9 system. But the CRISPR-Cas9 system has developed rapidly after the appearance of the other two technologies with its unique advantages. The CRISPR-Cas9 system is composed of clustered regularly spaced palindromic repeats and CRISPR-associated protein 9. The CRISPR-Cas9 system requires a sgRNA (small guide RNA, sgRNA) as a guide to recognize the target sequence and guide the Cas9 protein to complete its cleavage, causing a double-strand break to occur, which will trigger the endogenous repair mechanism. Endogenous repair mechanisms in cells include homologous recombination repair mechanism and non-hom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N9/22C12N15/09A01K67/027C12N5/10
CPCA01K67/0278A01K2227/102A01K2267/02C12N9/22C12N15/102C12N15/907
Inventor 刘东军郝斐闫玮
Owner INNER MONGOLIA UNIVERSITY
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