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CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method

An exogenous gene and gene knockout technology, which is applied in the fields of molecular biology and animal genetics and breeding, can solve problems such as unreported research, and achieve the effects of improving safety, reducing length, and improving integration efficiency

Active Publication Date: 2016-06-15
INNER MONGOLIA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Knockout of sheep MSTN gene with CRISPER-Cas9 system and at the same time integrated hfat-1 gene at the gRNA recognition site has not been reported

Method used

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  • CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method
  • CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method
  • CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The optimization of embodiment 1 CRISPER-Cas9 carrier

[0039] The CRISPR-Cas9 expression vector purchased from Beijing Zhongyuan Company was optimized. The nucleotide sequence of the optimized CRISPER-Cas9 vector (i.e. hCas9 plasmid) is shown in SEQ ID NO: 1, and the hCas9 plasmid map is shown in figure 1 .

Embodiment 2

[0040] The construction of embodiment 2gRNA expression vector

[0041] According to the sheep MSTN gene sequence (GeneID100860887), the gRNA sequence was designed for the exon 1 sequence of MSTN, 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.

[0042] Among them, the DNA sequence of the gRNA action site is as follows:

[0043] 5′-CGATGACTACCACGTTACGA-3′ (gRNA1 target site)

[0044] 5′-CGTTACGACGGAAACGGTCA-3′ (gRNA2 target site)

[0045] Use biological software to design gRNA sequences according to the gRNA action sites (gRNA1 target site and gRNA2 target site), clone them into the PMD-19T vector, transform Escherichia coli Trans-110, pick a single colony after plating, and perform bacterial Liquid PCR, identified by electrophoresis and sequencing, the single colony with correct sequencing was inoculated in LB medium containing...

Embodiment 3

[0046] Example 3 Efficiency Detection of CRISPER-Cas9 System

[0047] Premier5 software was used to design PCR primers spanning different target sites, and the genomes of sheep fibroblasts transfected with hCas9 plasmids and RNA1-MSTN / RNA2-MSTN48h plasmids were extracted, and the genomes were used as templates for PCR amplification. Primers are as follows:

[0048] MSTN-B-F: 5′-CTATTTATGCTGCTTGTTGC-3′

[0049] MSTN-B-R: 5′-CTATCTCCCAATCCTTCACC-3′

[0050] The total PCR reaction system is 50 μL, premixed ExTaq 25 μL, upstream and downstream primers (100 mmol L -1 ) each 2 μL, genomic DNA 2 μL, sterilized water 19 μL. PCR reaction conditions: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 1 min, annealing at 50°C for 30 s, extension at 72°C for 45 s, 35 cycles; 10 min at 72°C, 30 min at 16°C. The size of the amplified fragment is 666bp. The fragments containing gRNA1 and gRNA2 target sites were amplified from the sheep genome, detected by 1.5% agarose gel elec...

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Abstract

The invention relates to a CRISPER-Cas9-system-mediated sheep MSTN (myostatin) gene knock-out and exogenous gene site-specific integration method. The method comprises the following steps: establishing a CRISPER-Cas9-system-based gRNA (guide ribonucleic acid) expression vector according to a sheep MSTN gene sequence, establishing an exogenous-gene-containing donor plasmid capable of being integrated into a host genome according to the acting site of the gRNA, and transforming the optimized CRISPER-Cas9 vector, the gRNA expression vector and the linearized donor plasmid into sheep fibroblasts, thereby obtaining the sheep MSTN gene knock-out and exogenous gene site-specific integrated cells. The CRISPER-Cas9-mediated targeted vector provides a simple quick safe way for sheep MSTN gene knock-out and exogenous gene site-specific integration, thereby greatly enhancing the screening efficiency of the transgenic cell line. The method can screen the site-specific integrated exogenous gene cell line without adding any selection marker, thereby greatly enhancing the safety of the transgenic animals and having important value for sheep genetic breeding.

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 sheep MSTN gene mediated by a CRISPER-Cas9 system and for site-specific integration of foreign genes. Background technique [0002] The CRISPR-Cas9 gene editing system is composed of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). The genome editing technology developed rapidly after the factor-like effector nuclease (transcription activator-like effector nucleases, TALENs) technology has been widely used in cell gene knockout. CRISPR-Cas9 recognizes the target sequence through a small guide RNA (smallguideRNA, sgRNA) and guides the Cas9 protein to cut the target site, causing DNA double-strand break (DNA double-strandbreak, DSB), and the broken DNA will automatically start to prevent degradation Endogenous repair mechanism, and there are usually two kinds of end...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A01K67/027
CPCA01K67/0275A01K2217/072A01K2217/075A01K2227/102A01K2267/02C12N9/0071C12N15/8509C12N15/8772C12N2800/107C12N2800/60C12N2800/80C12N2810/10C12Y114/19004
Inventor 仓明张驹梁浩聂永伟梁宏宇崔梦兰刘东军
Owner INNER MONGOLIA UNIVERSITY
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