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CRISPR system for constructing double-gene combined knockout atherosclerotic porcine nuclear transplantation donor cells and application ofCRISPR system

A gene and gene editing technology, applied in the biological field, can solve the problems of not being able to truly simulate human physiology, pathology, mouse body size, organ size, physiology, and pathology, and achieve low and high costs for cloning and breeding , the effect of high feeding cost

Active Publication Date: 2021-06-29
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the commonly used animal model is the mouse model, but mice are greatly different from humans in terms of body shape, organ size, physiology, and pathology, and cannot truly simulate the normal physiological and pathological states of humans.

Method used

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  • CRISPR system for constructing double-gene combined knockout atherosclerotic porcine nuclear transplantation donor cells and application ofCRISPR system
  • CRISPR system for constructing double-gene combined knockout atherosclerotic porcine nuclear transplantation donor cells and application ofCRISPR system
  • CRISPR system for constructing double-gene combined knockout atherosclerotic porcine nuclear transplantation donor cells and application ofCRISPR system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, the preparation of plasmid

[0088] 1.1 Preparation of plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (plasmid pKG-GE3 for short)

[0089] The sequence of the original plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (abbreviated as plasmid pX330) is shown in SEQ ID NO:1. The schematic diagram of the structure of plasmid pX330 is shown infigure 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).

[0090] Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO ( Figure 5 ), referred to as plasmid pKG-GE3, the nucleotide is shown in SEQ ID NO:2. Compared with the plasmid pX330, the plasmid pKG-GE3 has been mainly modified as follows: ① Remove the residual gRNA backbone sequence...

Embodiment 2

[0103] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0104] Select high-efficiency gRNA targets located at the RAG1 gene:

[0105] Target of RAG1-gRNA4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 9).

[0106] Primers used to amplify and detect target-containing fragments are as follows:

[0107] RAG1-nF126: 5'-CCCCATCCAAAGTTTTTAAAGGA-3' (SEQ ID NO: 10);

[0108] RAG1-nR525: 5'-TGTGGCAGATGTCACAGTTTAGG-3' (SEQ ID NO: 11)

[0109] Primary porcine fibroblasts were prepared from the ear tissue of newborn Congjiang pigs (female, blood type AO).

[0110] 1. Preparation of recombinant plasmids

[0111] 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. RAG1-4S and RAG1-4A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with cohesive ends. The double-stranded DNA molecule with cohesive ends and the vector ...

Embodiment 3

[0134] Example 3, Target Screening for APOE Gene Knockout

[0135] Pig APOE gene information: encodes apolipoprotein E protein; located on pig chromosome 6;

[0136] GeneID is 397576, Sus scrofa. The protein encoded by the porcine APOE gene is shown in GENBANK ACCESSIONNO.NP_999473.1 (linear CON 12-JAN-2018). In the genomic DNA, the porcine APOE gene has 3 exons, wherein the second exon and its upstream and downstream 400bp sequences are shown in SEQ ID NO: 14, and its encoded protein fragment is shown in SEQ ID NO: 15.

[0137] 1. APOE gene knockout preset target and adjacent genome sequence conservation analysis

[0138] 18 newborn Congjiang pigs, including 10 females (named 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) and 8 males (named A, B, C, D , E, F, G, H).

[0139] Using the genomic DNA of 18 pigs as templates, PCR amplification was carried out using primer pairs (the target sequence of the primer pairs includes exon 2 of the porcine APOE gene), and then electrophoresis was per...

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PUM

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Abstract

The invention discloses a CRISPR / Cas9 system forporcine APOE and LDLR gene editing. The CRISPR / Cas9 system comprises a Cas9 expression vector and a gRNA expression vector aiming at porcine APOE and LDLR genes, wherein the Cas9 expression vector is a pU6gRNA-eEF1a-mNLS-hSpCas9-EGFP-PURO vector with the plasmid complete sequence shown in SEQ ID NO.2. A CRISPR / Cas9 gene editing technology is adopted, APOE and LDLR genes of pigs are knocked out, and the APOE and LDLR gene double-knocked-out pig recombinant cell is prepared, so that a solid foundation is laid for further production of atherosclerotic cloned pigs through a somatic cell cloning technology, and a powerful experimental tool is provided for treatment of atherosclerotic diseases and research on pathogenesis of the atherosclerotic diseases.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR / Cas9 system for gene editing of APOE and LDLR and its application in constructing atherosclerotic pig nuclear transplantation donor cells. Background technique [0002] Cardiovascular disease is currently the leading cause of death among all diseases among residents in our country. According to reports, the death rate of cardiovascular disease accounts for more than 40% of the residents' disease deaths, which is much higher than that of cancer and other diseases. Atherosclerosis is the main cause of coronary heart disease, cerebral infarction and peripheral vascular disease. Abnormal lipid metabolism and inflammatory response are the main lesion basis of atherosclerosis. [0003] Apolipoprotein E (APOE) is a major prosthetic protein of chylomicrons, which can bind to receptors on liver cells or surrounding cells. Defects in the APOE gene may lead to elevated se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/12C12N5/10C12N15/877A01K67/027
CPCC07K14/47C12N15/8509C12N15/8778A01K67/0276A01K2227/108A01K2267/0375A01K2217/075C12N2310/20Y02A50/30
Inventor 牛冬汪滔马翔刘璐曾为俊王磊程锐赵泽英陶裴裴黄彩云
Owner NANJING KGENE GENETIC ENG CO LTD
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