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SgRNA targeting sequence of specific target pig MC4R gene and applications of sgRNA targeting sequence

A variety of specific and genetic technologies, applied in sgRNA guide sequences and application fields, can solve the problems of inability to obtain non-homologous terminal mutations, and achieve high safety and eliminate expression effects

Inactive Publication Date: 2017-09-01
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cas9 proteins with mutations in the RuvC domain can also be repaired by homologous recombination, but non-homologous end-joining mutations cannot be obtained

Method used

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  • SgRNA targeting sequence of specific target pig MC4R gene and applications of sgRNA targeting sequence
  • SgRNA targeting sequence of specific target pig MC4R gene and applications of sgRNA targeting sequence

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Experimental program
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Effect test

Embodiment 1

[0027] (1) sgRNA design

[0028] According to the genome sequence of porcine MC4R gene (gene ID: NM214173), a sgRNA targeting porcine MC4R gene was designed. The 20nt oligonucleotide sgRNA guide sequence is: MC4R-sgRNA1: 5'-TGTGCAGTCCGTAGGTGCTG-3', located in the exon coding region of the gene MC4R; add CACC to its 5' end to obtain the forward oligonucleotide sequence; Obtain its corresponding DNA complementary strand according to the guide sequence, and add AAAC to its 5' end to obtain a reverse oligonucleotide. Synthesize the above forward oligonucleotides and reverse oligonucleotides respectively, and synthesize the forward and reverse sgRNA oligonucleotide sequences: denature at 95°C for 5 minutes, and anneal at 72°C for 10 minutes; after annealing, sticky ends can be formed The specific oligonucleotide sequence is shown in Table 1.

[0029] Table 1 Oligonucleotide sequence of sgRNA guide sequence

[0030] Nucleotide sequence (5' to 3') Sg1-F CACCTGTG...

Embodiment 2

[0036] (1) Culture and cryopreservation of porcine fibroblasts

[0037] Put the pig fetus in a large domestic dish and add physiological saline to wash it 5-6 times. After washing, remove the fetal limbs, tail, head and internal organs. Put the processed fetus into an imported small dish, cut it into pieces with scissors, and terminate it until the largest tissue piece is 1mm3. Add 2ml of 0.25% EDTA-Trypsin to the small dish, place the small dish in a 37 degree incubator for 25 minutes to digest, then stop the digestion with 4ml of culture medium DMEM / F12 (10% FBS), blow it with a Pasteur pipette and suck out the liquid In a 15ml centrifuge tube, centrifuge at 1000rpm for 3min. After centrifugation, discard the supernatant, add 3ml of culture medium to resuspend, and divide evenly among three imported large dishes, and add 8ml of culture medium DMEM / F12 (10% FBS) to the imported large dishes. Observe the state of the cells the next day, and if the cell volume reaches 90%, pa...

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Abstract

The invention relates to a sgRNA targeting sequence of specific target pig MC4R gene and applications of the sgRNA targeting sequence, relating to a gRNA targeting sequence and applications. The sgRNA targeting sequence is CGTCTCGCGCTTGGACTCAG. The sgRNA targeting sequence can knock out or edit MC4R gene through a CRISPR / Cas9 system, and further eliminate the expression of MC4R, thus providing basis for preparing MC4R transgenic pigs. The sgRNA targeting sequence is applied to the field of genetic engineering.

Description

technical field [0001] The invention relates to a sgRNA guide sequence and its application. Background technique [0002] In the past decade, a new research method has emerged that can help researchers artificially manipulate almost any gene in various cells and various organisms. This new technology is what we often call "genome editing technology". Both ZFN and TALEN can perform various genetic modifications on DNA. The mechanism of action of these two nucleases is to first cut DNA double-stranded molecules to form DNA double The strand-breaking nick then activates the intracellular non-homologous end-joining repair mechanism, or the homologous recombination repair mechanism, which uses the cell's own repair mechanism to genetically modify the DNA. The latest CRISPR / Cas9 gene editing system has been greatly developed and applied once it appeared. The CRISPR / cas9 gene editing system is modified based on the class II CRISPR / Cas system. Cas9 protein is the only Cas protein...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/90A01K67/027
CPCC12N15/113A01K67/0276A01K2207/15A01K2217/075A01K2227/108A01K2267/03C07K14/47C12N15/8509C12N15/907
Inventor 牟彦双刘忠华郭佳马伟超郭超
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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