Targeting sgRNA for pig CD163 gene editing, modification vector, and preparation method and application thereof

A gene modification and gene editing technology, applied in the field of genetic engineering, to ensure normal survival, reduce the work of genetically modified safety assessment, and have strong specific effects

Inactive Publication Date: 2017-09-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although more and more genes are currently being used as biomarkers and therapeutic targets for livestock diseases, the key genes in pig PRRS and their editing and modification methods have not yet been applied in my country

Method used

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  • Targeting sgRNA for pig CD163 gene editing, modification vector, and preparation method and application thereof
  • Targeting sgRNA for pig CD163 gene editing, modification vector, and preparation method and application thereof
  • Targeting sgRNA for pig CD163 gene editing, modification vector, and preparation method and application thereof

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preparation example Construction

[0050] The present invention also provides a preparation method for the above-mentioned porcine CD163 gene modification carrier, comprising the following steps:

[0051] Step (a): Complementary pairing of the sense strand and the antisense strand of sgRNA-C161 to form a C161 double-stranded DNA molecule;

[0052] Step (b): Complementary pairing of the sense strand and the antisense strand of sgRNA-C185 to form a C185 double-stranded DNA molecule;

[0053] Step (c): connecting the C161 double-stranded DNA molecule to an expression vector to construct a vector expressing sgRNA-C161;

[0054] Step (d): connecting the C185 double-stranded DNA molecule to an expression vector to construct a vector expressing sgRNA-C185;

[0055] Step (e): Using the vector expressing sgRNA-C161 as a template, amplify the sgRNA-C161 sequence containing the U6 promoter by PCR, connect it to the cloning vector, and obtain the U6-sgRNA-C161 sequence after enzyme digestion;

[0056] Step (f): link the ...

Embodiment 1

[0072] The design of embodiment 1sgRNA and the construction of carrier

[0073] According to the seventh exon, the eighth exon and part of the upstream and downstream intron sequences (as shown in SEQ ID NO.5) and the mRNA sequence (NCBI NM_213976.1, as shown in SEQ ID NO.6) of the pig CD163 gene ), designing two sgRNAs on the seventh exon of the CD163 gene, sgRNA-C161 and sgRNA-C185, respectively adding cohesive linker sequences to both ends. Add the adapter sequence CACC to the 5' end of the F strand of each sgRNA sequence, and add the adapter sequence AAAC to the 5' end of the R strand of the reverse complementary sequence. If the first base at the 5' end of the sgRNA sequence is not G, then it should be First add a G to the 5' end of the F chain of the sgRNA sequence, and then add the linker sequence CACC. Correspondingly, add a C to the 3' end of the R chain of the reverse complementary sequence, so that it can be combined with the PX461 vector digested by BbsI The cohes...

Embodiment 2

[0101] Example 2 Transfection of pig somatic cells and screening of CD163 gene edited cell clones

[0102] Cell culture and transient transfection:

[0103] Pig fetal fibroblasts were inoculated in 6-well cell culture, and when they were cultivated to 50-70% confluence in DMEM medium containing 15% fetal bovine serum, according to the requirements of the instructions, Lipofectamine 2000 liposomes were provided in Example 1. The vector PX461-C185+C161 was transferred into the cells.

[0104] Flow cytometric sorting and monoclonal culture of transfected cells:

[0105] The transfected cells were cultured in a 37°C incubator for 48 hours, digested with 0.1% trypsin, sorted the positive cells expressing GFP by flow cytometry, and seeded the cells at a density of 50-100 cells / 100mm culture dish , cultured in DMEM medium containing 15% fetal bovine serum and 2.5ng / mL fibroblast growth factor (bFGF) for 9 to 12 days until obvious single-cell colonies appeared.

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Abstract

The invention provides a targeting sgRNA for pig CD163 gene editing, a modification vector, and a preparation method and application thereof, which relate to the technical field of gene engineering. The sgRNA and the gene modification vector provided by the invention have high specificity, and can be used for efficiently editing a pig CD163 gene on a cell level through a CRISPR/Cas9n system. According to the preparation method of a blue-eared disease resistant pig provided by the invention, a blue-eared disease virus receptor is damaged, the target gene CD163 is edited, no other exogenous gene is intruded, no non-specific editing is carried out on a non-CD163 gene area on a genome, a genetic background is clean and clear, and the work of later transgene safety evaluation is greatly reduced. The blue-eared disease resistant pig obtained by utilizing the invention can be ensured to survive normally, and resists the infection of a blue-eared disease virus, so that the economic loss caused by the blue-eared disease in pig husbandry is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a targeting sgRNA for porcine CD163 gene editing, a modified carrier, a preparation method and application thereof. Background technique [0002] Porcine blue ear disease (porcine reproductive and respiratory syndrome, PRRS) is the first major viral infectious disease that seriously harms the pig industry, and brings billions of dollars of losses to the global pig industry every year. Pig blue-ear disease is caused by porcine reproductive and respiratory syndrome virus (PRRSV), which mainly affects pregnant sows, piglets and breeding boars, causing premature birth, abortion and stillbirth in pregnant sows, breathing difficulties and even death in piglets. [0003] Foreign studies have shown that the protein region encoded by the seventh exon of the porcine CD163 gene is the region where the PRRS virus receptor binds to the PRRS virus, and the CD163 gene of the seventh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22A01K67/027
CPCC12N15/1131A01K67/0276A01K2217/075A01K2227/108A01K2267/02C12N15/907
Inventor 张坤王少华
Owner ZHEJIANG UNIV
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