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Application of CRISPR/Cas9 carrier combination in preparation of blood product of gene knockout pig

A GGTA1-CRISPR, gene knockout technology, applied in the field of genetic engineering, to achieve the effect of a source of precious materials, solving clinical ischemia, and overcoming hyperacute immune rejection

Pending Publication Date: 2018-09-28
GCREATENE SUZHOU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Purpose of the invention: In order to solve the immune rejection existing in the clinical transfusion of heterologous red blood cells, the present invention provides the application of a combination of CRISPR / Cas9 vectors in the preparation of blood products for knockout pigs

Method used

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  • Application of CRISPR/Cas9 carrier combination in preparation of blood product of gene knockout pig
  • Application of CRISPR/Cas9 carrier combination in preparation of blood product of gene knockout pig
  • Application of CRISPR/Cas9 carrier combination in preparation of blood product of gene knockout pig

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of CRISPR / Cas9 vector

[0036] First, according to the DNA sequence of the GGTA1 / CMAH / β4GalNT2 gene, sgRNA (single guide RNA) targeting GGTA1, CMAH and β4GalNT2 genes were synthesized, and pX330 was used as the backbone plasmid to construct the GGTA1-CRISPR / Cas9 vector and the CMAH-CRISPR / Cas9 vector respectively and β4GalNT2-CRISPR / Cas9 vector.

[0037] 1. The GGTA1-CRISPR / Cas9 vector is prepared as follows:

[0038] First, according to the porcine GGTA1 gene sequence published in Genbank, exon 3 of the GGTA1 gene was selected as the CRISPR / Cas9 target. Design the sgRNA sequence as GAAAATAATGAATGTCAA, see figure 1 , the nucleotide sequence is shown in SEQ ID No:1.

[0039] The GGTA1-CRISPR / Cas9 vector was prepared as follows:

[0040] Step 1. According to the principle of cas9 target design: G at the 5' end and PAM sequence (NGG) at the 3' end, find the target position on the GGTA1 gene;

[0041] Step 2. Purchase the pX330 backbone plasmid (...

Embodiment 2

[0122] Example 2 Construction of GGTA1 / CMAH / β4GalNT2 Three Gene Knockout Pigs by Somatic Cell Cloning

[0123] The constructed GGTA1-CRISPR / Cas9 vector, CMAH-CRISPR / Cas9 vector and β4GalNT2-CRISPR / Cas9 vector were co-transfected into porcine fetal fibroblasts with tdTomato plasmid. Single-cell clones were obtained by G418 screening, GGTA1 / CMAH / β4GalNT2 triple gene knockout pig fetal fibroblasts were obtained by sequencing, and GGTA1 / CMAH / β4GalNT2 triple gene knockout Landrace pigs were prepared by somatic cell nuclear transfer (SCNT). The genome of newborn piglets was extracted, amplified with PCR primers, and connected with T vectors for genotype identification.

[0124] Step 1. Recovery of porcine primary fibroblasts

[0125] 1. Take out the frozen primary porcine fibroblasts from liquid nitrogen, and thaw them in a 37°C water bath;

[0126] 2. Transfer the thawed cells into a sterile 15mL centrifuge tube, then add 3mL cell culture medium, and centrifuge at 1500rpm for 5mi...

Embodiment 3

[0187] Example 3 Phenotype analysis of GGTA1 / CMAH / β4GalNT2 triple gene knockout pigs

[0188] 1. Knockout of GGTA1, CMAH and β4GalNT2 genes in wild-type pigs can effectively reduce hyperacute immune rejection during xenotransplantation

[0189] After the piglets were weaned, blood was drawn, peripheral blood mononuclear cells (PBMC) were separated, and the gene knockout status of the piglets was measured by flow cytometry, as well as the combination with immunoglobulins (IgM, IgG) in human serum. It was found that α-1,3-galactosyltransferase (GGTA1), CMP-N-acetylneuraminic acid hydroxylase (CMAH) and β-1,4-N of the three gene knockout piglets prepared in Example 2 - Three antigens of acetylgalactosaminyl transferase 2 (β4GalNT2) were successfully knocked out, such as Image 6 As shown, where PBSControl is blank control, Isotype Control is chicken IgY, WT is wild type pig, GGTA1-KO is GGTA1 gene knockout pig, CMAH-KO is CMAH gene knockout pig, β4GalNT2-KO is β4GalNT2 gene knoc...

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Abstract

The invention discloses the application of a CRISPR / Cas9 carrier combination in preparation of a blood product of a gene knockout pig. The gene knockout pig is a pig without GGTA1 genes, CMAH genes and beta4Ga1NT2 genes. The CRISPR / Cas9 carrier combination comprises a GGTA1-CRISPR / Cas9 carrier, a CMAH-CRISPR / Cas9 carrier and a beta4Ga1NT2-CRISPR / Cas9. By designing a specifically targeted SgRNA sequence, the knockout efficiency of the three genes is respectively 56 percent, 63 percent and 41 percent; after the three genes relating to immunological rejection are knocked out, the gene knockout pig is obtained; the combination between erythrocytes and immune globulin in human serum is obviously reduced; an outstanding effect is achieved to overcome hyperacute immunological rejection; the problem of clinical ischemia is effectively solved; a precious material source is provided for clinical blood transfusion.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of a CRISPR / Cas9 carrier combination in preparing blood products of gene knockout pigs. Background technique [0002] With the continuous advancement of modern medicine, blood transfusion is widely used clinically, but it also faces more and more problems, including high-incidence infectious diseases such as AIDS, hepatitis B, and hepatitis C, which lead to serious risks of blood transfusion. The demand for blood continues to increase while the amount of donated blood is relatively reduced, and blood resources are becoming increasingly scarce. From the 19th century to the present, xenotransfusion research has been devoted to developing a substitute for human RBCs transfusion with animal red blood cells (RBCs). In the event of acute blood loss in accidents or war situations, xenogeneic blood transfusion can maintain physiological functions ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/873C12N15/66C12N5/10
CPCC12N5/0641C12N9/0073C12N9/1051C12N9/22C12N15/66C12N15/85C12N15/873C12N15/907C12Y204/01041C12N2510/00C12N2800/107C12N2810/10C12N5/0634C12N2310/20C12Y204/01087C12Y204/01165C12Y114/18002C12N9/0071A01K2227/108A01K2267/025A01K2217/075A01K67/0276A61K35/14A61K35/18C12N15/8509C12N2800/80
Inventor 戴一凡杨海元王盈
Owner GCREATENE SUZHOU BIOTECH CO LTD
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