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A method for preparing CD163 gene-edited pigs using the spry-cas9 system

A gene editing and gene technology, which is applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of reducing off-target effects, making it difficult for researchers to design and edit candidate gene regions, and off-target problems

Active Publication Date: 2021-05-11
北京首农未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, this technology currently has two main defects: first, there is off-target phenomenon. At present, the off-target effect is greatly reduced by the upgraded nickase version of dCas9, high-fidelity Cas9, dCas9-forkI and gRNA modification.
Second, the limitations of PAM make it difficult for researchers to freely design and edit candidate gene regions

Method used

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  • A method for preparing CD163 gene-edited pigs using the spry-cas9 system
  • A method for preparing CD163 gene-edited pigs using the spry-cas9 system
  • A method for preparing CD163 gene-edited pigs using the spry-cas9 system

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Embodiment 1

[0060]Example 1, using SPRY-Cas9 system to prepare CD163 bis allele mutation cell lines

[0061]This embodiment uses the SPRY-Cas9 system to prepare a CD163 double allele mutation cell line. Considering the latter industrialization, avoiding biological safety issues, the present invention does not use a screening marker gene while using a Cas9 protein mRNA, which can prepare a CD163 double allele mutation cell line of an absence of external DNA integration, technical routefigure 1 .

[0062]First, the substance used for CD163 double allele mutation

[0063]1, express vector spry-sgRNA

[0064]In this study, the exon 3 region of the CD163 gene was selected as the target site, and the sequences of the CD163 gene were as shown in the sequence 1, as shown in Sequence 2.

[0065]3 GRNAs were designed to identify sequences (target sequences of SGRNA) were: GRNA-1: atgtttcttgtcgaggaat; GRNA-2: Gatgatgatgtgatgatgtgtc; GRNA-3: GatcatgttctTgtcgagg.

[0066]Single-stranded oligonucleotides are synthesized accor...

Embodiment 2

[0157]Example 2, CD163 double allele knockout pig preparation

[0158]I. Preparation of CD163 double allele knockout pig

[0159]1. Take the CD163 bis-allele pig fibroblast mutant obtained in the long-term long-term long-term period of time, and 0.25% trypsin is digested for 5 min to give a single cell, and subsequent operation is donor cells.

[0160]2, from the slaughter plant, the ovary of the adult white pig, washed three times after 37 ° C in the PBS liquid, with a diameter of 0.7mm needle to extract a diameter of 2-8 mm, and the form is uniform, and the structural dense cubo-oocyte Cell-Complex (Cocs), washed twice with ripe solution (M199 + 10% FBS + 0.01 U / mL BFSH + 0.01 U / mL BLH + 1 μg / ml estradiol), then cooled - oocytes - The complex is placed in a ripe solution with a mature liquid in 50-60 / wells, at 38.5 ° C, 5% CO2After 18-20 h in the incubator, the mature oocytes were oscillated with 0.1% hyaluronidase. After 2-3 minutes of the tube of hyaluronase, it was gently blown ...

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Abstract

The invention discloses a method for preparing CD163 gene-edited pigs by using the SpRY-Cas9 system. The present invention provides a method for preparing CD163 biallelic mutant cells. In order to genetically edit the CD163 gene of the porcine fibroblast genome in vitro, the 226- The GTCGAGGGAATGA at position 238 is deleted, the GTCGAGGGAAT at position 226-236 of the third exon of another chromosome is deleted, and the other nucleotide sequences remain unchanged, so that CD163 biallelic mutant cells are obtained; then the cells are used to prepare CD163 biallelic Gene knockout pigs have the ability to resist PRRSV virus, indicating that the method for preparing CD163 biallelic knockout pigs of the present invention can obtain pigs resistant to PRRSV virus.

Description

Technical field[0001]The present invention belongs to the field of biotechnology, and involves a method of editing a pig using a SPRY-Cas9 system.Background technique[0002]Porcine Reproductive and Respiratory Syndrome, PRRS is a global, intrinsic, harmful porcine toxicity, which is different in all ages of pigs, but mainly Leading the advanced abortion, dead and mummies in the pregnant sow, and the pig's respiratory disease, with high mortality, major economic losses in the global pig industry, such as in the United States, PRRS total economic losses per year is about 6.64 US dollars, causing the loss of 1.5 billion euros in Europe, probably caused a loss of 7 billion yuan in China.[0003]The disease was first burst in the United States in 1987, followed by rapid spreading to the world in 1990-1992. Its pathogens is pig breeding and respiratory syndrome virus (PRRSV), which mainly has two types I and II. PRRSV is a single-shaped just RNA virus, a member of Nidovirales, ArteriRiRidae,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N5/10C12N15/12A01K67/027
CPCA01K67/0275A01K2227/108A01K2267/02C07K14/70596C12N15/85
Inventor 沈秋平李垲孙照霖
Owner 北京首农未来生物科技有限公司
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