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CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects

A technology for recombining cells and fused genes, applied to cells modified by introducing foreign genetic material, genetic engineering, recombinant DNA technology, etc., can solve problems such as muscle necrosis, cell death, and loss of muscle tissue function

Pending Publication Date: 2021-03-19
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pathologically, Duchenne muscular dystrophy is due to changes in the structure and function of dystrophin (also known as dystrophin, dystrophin in English), resulting in progressive degeneration, necrosis, and pseudohypertrophy of the calf gastrocnemius muscle in the proximal extremities. , resulting in muscle necrosis and cell death; although the surrounding satellite cells are activated to form muscle cell regeneration, the regeneration of muscle cells will eventually stop due to the aging of satellite cells, and the muscle tissue will eventually be infiltrated by fibrous fat and lose muscle tissue Function
There are no reports of large animal models of DMD

Method used

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  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects
  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects
  • CRISPR/Cas9 system and application thereof in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, the preparation of plasmid

[0076] The plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 was prepared, as shown in SEQ ID NO:1. Plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9, referred to as plasmid pX330.

[0077] The plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO was prepared, as shown in SEQ ID NO:2. Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3.

[0078] The plasmid pKG-U6gRNA was prepared, as shown in SEQ ID NO:3.

[0079] Plasmid pX330, plasmid pKG-GE3, and plasmid pKG-U6gRNA are all circular plasmids.

[0080] The schematic diagram of the structure of plasmid pX330 is shown in figure 1 . In SEQ ID NO: 1, the 440-725 nucleotides form the CMV enhancer, the 727-1208 nucleotides form the chickenβ-actin promoter, and the 1304-1324 nucleotides encode the SV40 nuclear localization signal (NLS ), the 1325-5449th nucleotide encodes the Cas9 protein, and the 5450-5497th nucleotide encodes the nucleoplasmin nuclear localization signal (NLS...

Embodiment 2

[0084] Example 2, Plasmid Proportion Optimization and Effect Comparison of Plasmid pX330 and Plasmid pKG-GE3

[0085] Select two gRNA targets located at the MSTN gene:

[0086] Target of MSTN-gRNA1: 5'-GCTGATTGTTGCTGGTCCCG-3';

[0087] Target of MSTN-gRNA2: 5'-TTTCCAGGCGAAGTTTACTG-3'.

[0088] Select two gRNA targets located on the FNDC5 gene:

[0089] Target of FNDC5-gRNA1: 5'-TGTACTCAGTGTCCTCCTCC-3';

[0090] Target of FNDC5-gRNA2: 5'-GCTCTTCAAGACGCCTCGCG-3'.

[0091] Primers used to amplify target-containing fragments are:

[0092] MSTN-F896: 5'-TCTCTCAGACAGTGCAGGCATTA-3';

[0093] MSTN-R1351: 5'-CGTTTCCGTCGTAGCGTGATAAT-3'.

[0094] FNDC5-F209: 5'-CAGTTCTCACTTGATGGCCTTGG-3';

[0095] FNDC5-R718: 5'-AGGGGTCTGGGGAGGAATGG-3'.

[0096] 1. Preparation of recombinant plasmids

[0097] The plasmid pKG-U6gRNA was taken, digested with restriction endonuclease BbsI, and the vector backbone (a large linear fragment of about 3 kb) was recovered.

[0098] MSTN-1S and MSTN-1A w...

Embodiment 3

[0135] Embodiment 3, the screening of the target combination of DMD gene knockout

[0136] 1. DMD gene knockout preset target and adjacent genome sequence conservation analysis

[0137] Pig DMD gene information: encoding dystrophin; located on the X chromosome; GeneID is 1756, Sus scrofa. The protein encoded by the porcine DMD gene is shown in SEQ ID NO:4. In the genomic DNA, the porcine DMD gene has 89 exons, wherein the 46th exon and its upstream and downstream sequences of 500 bp are shown in SEQ ID NO:5.

[0138] Genomic DNA of 8 pigs was respectively used as templates, and primer pairs (the target sequence of the primer pairs included exon 46 of porcine DMD gene) were used for PCR amplification, and then electrophoresis was performed. The PCR amplification products were recovered and sequenced, and the sequencing results were compared with the DMD gene sequence in the public database. According to the comparison results, primers for detecting mutations were designed (t...

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Abstract

The invention discloses a CRISPR / Cas9 system and application of the CRISPR / Cas9 system in construction of swine-derived recombinant cells for resisting amyotrophy protein gene defects. The invention provides an sgRNA combination, the sgRNA combination is composed of sgRNADMD-Ug3 (the target sequence binding region is shown as the 1-20th nucleotide in SEQ ID NO: 8) and sgRNADMD-Dg3 (the target sequence binding region is shown as the 1-20th nucleotide in SEQ ID NO: 11). The invention provides a kit, the kit is composed of a plasmid pKG-U6gRNA (DMD-Ug3) of sgRNADMD-Ug3 obtained through transcription, a plasmid pKGU6gRNA (DMD-Dg3) of sgRNADMD-Ug3 obtained through transcription, and a plasmid pKG-GE3. The sgRNA combination or the kit can be used for preparing recombinant cells or preparing a Duchenne muscular dystrophy animal model. The recombinant cells are anti-amyotrophy protein gene defect cells and can be used for preparing an animal disease model through somatic cell cloning. A solidfoundation is laid for the preparation of a Duchenne muscular dystrophy swine model, and has important application value for the research and development of Duchenne muscular dystrophy medicines.

Description

technical field [0001] The invention relates to a CRISPR / Cas9 system and its application in the construction of pig-derived recombinant cells with dystrophin gene defects. Background technique [0002] Duchenne muscular dystrophy (DMD) is an X-chromosome recessive genetic disease. Because the disease is an X-linked recessive genetic disease, the main patients of this disease are males, and the incidence rate of males is about 1 / 3500-1 / 5000, and 1 / 3 of the patients are caused by autologous gene mutations. Most women are carriers and usually do not develop the disease. Pathologically, Duchenne muscular dystrophy is due to changes in the structure and function of dystrophin (also known as dystrophin, dystrophin in English), resulting in progressive degeneration, necrosis, and pseudohypertrophy of the calf gastrocnemius muscle in the proximal extremities. , resulting in muscle necrosis and cell death; although the surrounding satellite cells are activated to form muscle cell r...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N5/10A01K67/027
CPCC12N15/113C12N15/8509C07K14/4708A01K67/0276C12N2310/20A01K2207/15A01K2217/075A01K2227/108A01K2267/0306Y02A50/30
Inventor 牛冬汪滔马翔曾为俊王磊程锐赵泽英
Owner NANJING KGENE GENETIC ENG CO LTD
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