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CRISPR system for constructing MC4R gene mutated obesity porcine nuclear transfer donor cell and application of CRISPR system

A gene and gene editing technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, receptors/cell surface antigens/cell surface determinants, etc., can solve the problems of susceptibility differences and reduce work Quantity, good applicability

Active Publication Date: 2021-04-06
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although obesity is usually influenced by both genetics and the environment, obesity is actually heritable and polygenic, but the susceptibility of different people varies

Method used

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  • CRISPR system for constructing MC4R gene mutated obesity porcine nuclear transfer donor cell and application of CRISPR system
  • CRISPR system for constructing MC4R gene mutated obesity porcine nuclear transfer donor cell and application of CRISPR system
  • CRISPR system for constructing MC4R gene mutated obesity porcine nuclear transfer donor cell and application of CRISPR system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1, the construction of plasmid

[0079] 1.1 Construction of plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (plasmid pKG-GE3 for short)

[0080] 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 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).

[0081] 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 sequ...

Embodiment 2

[0094] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0095] Select high-efficiency gRNA targets located at the RAG1 gene:

[0096] Target of RAG1-gRNA4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 9).

[0097] Primers used to amplify and detect target-containing fragments are as follows:

[0098] RAG1-nF126: 5'-CCCCATCCAAAGTTTTTAAAGGA-3' (SEQ ID NO: 10);

[0099] RAG1-nR525: 5'-TGTGGCAGATGTCACAGTTTAGG-3' (SEQ ID NO: 11)

[0100] Primary porcine fibroblasts were prepared from the ear tissue of newborn Congjiang pigs (female, blood type AO).

[0101] 1. Preparation of recombinant plasmids

[0102] 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

[0124] Example 3, Target Screening for MC4R Gene Knockout

[0125] Pig MC4R gene information: encodes melanocortin 4 receptor protein; located on pig chromosome 1; GeneID is 397359, Sus scrofa. The protein encoded by the pig MC4R gene is shown in GENBANK ACCESSION NO.NP_999338.1 (linear CON 12-JAN-2018). In the genomic DNA, the porcine MC4R gene has one exon, wherein the first exon is shown in SEQ ID NO: 14, and its encoded protein fragment is shown in SEQ ID NO: 15. 1. MC4R gene knockout preset target and adjacent genome sequence conservation analysis

[0126] 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).

[0127] Genomic DNA of 18 pigs was used as a template, and primer pairs (the target sequence of the primer pair includes the 12th exon of the pig MC4R gene) were used for PCR amplification, and then electrophoresis was performed. The PCR amplification products were recovered and sequenc...

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Abstract

The invention discloses a CRISPR system for constructing an MC4R gene mutated obesity porcine nuclear transfer donor cell and application of the CRISPR system. The system comprises a Cas9 expression vector and a gRNA expression vector for a porcine MC4R gene, wherein the Cas9 expression vector is a plasmid with a complete sequence as shown in SEQ ID NO.2, and the gRNA expression vector expresses gRNA as shown in SEQ ID NO.22; and the vector framework of the expression vector is pKG-U6gRNA, and the complete sequence of the plasmid is shown as SEQ ID NO.3. Gene editing is carried out by adopting the gRNA combined modified Cas9 high-efficiency expression vector screened by the invention, and the editing efficiency is remarkably improved compared with that of an original vector.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR / Cas9 system for MC4R gene editing and an application thereof. Background technique [0002] Obesity is a physical condition in which excess body fat accumulates and negatively affects health, which may lead to shortened life span and various health problems. Obesity is the leading preventable cause of death worldwide and one of the most important public health problems of the 21st century. The prevalence of obesity is increasing in both adults and children, and occurs more frequently in women than men. In 2013, several medical societies, including the American Medical Association and the American Heart Association, defined obesity as a disease, known as obesity. In 2015, 600 million adults (13%) and 42 million children under five were obese globally. The World Health Organization even issued a warning, pointing out that overweight and obesity are the fifth lar...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/113C12N5/10A01K67/027
CPCC12N15/8509C12N15/65C12N15/1138C12N9/22C07K14/72C12N5/0656A01K67/0276C12N2310/20C12N2800/107A01K2227/108A01K2267/0362C12N2510/00C12N2517/02C07K2319/09C12N2830/48C12N2830/36
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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