CRISPR system for ADCY3 gene editing and application of CRISPR system in construction of obese porcine nuclear transplantation donor cells

A gene editing and gene technology, applied in cells modified by introducing foreign genetic material, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve problems such as differences in susceptibility, and achieve the effect of reducing workload and improving application.

Active 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

[0003] Although obesity is usually influenced by both genetics and the environment, o...

Method used

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  • CRISPR system for ADCY3 gene editing and application of CRISPR system in construction of obese porcine nuclear transplantation donor cells
  • CRISPR system for ADCY3 gene editing and application of CRISPR system in construction of obese porcine nuclear transplantation donor cells
  • CRISPR system for ADCY3 gene editing and application of CRISPR system in construction of obese porcine nuclear transplantation donor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Embodiment 1, the preparation of plasmid

[0078] 1.1 Preparation of plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (plasmid pKG-GE3 for short)

[0079] 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).

[0080] Plasmid pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, referred to as plasmid pKG-GE3, the nucleotide is shown in SEQ ID NO:2. The build method is as follows:

[0081] (1) Remove redundant and invalid sequences in the gRNA backbone

[0082] Plasmid pX330 was digested with...

Embodiment 2

[0093] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0094] Select high-efficiency gRNA targets located at the RAG1 gene:

[0095] Target of RAG1-gRNA4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO: 9).

[0096] Primers used to amplify and detect target-containing fragments are as follows:

[0097] RAG1-nF126: 5'-CCCCATCCAAAGTTTTTAAAGGA-3' (SEQ ID NO: 10);

[0098] RAG1-nR525: 5'-TGTGGCAGATGTCACAGTTTAGG-3' (SEQ ID NO: 11)

[0099] Primary pig fibroblasts were prepared from newborn Congjiangxiang pigs (female, blood type AO).

[0100] 1. Preparation of recombinant plasmids

[0101] 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 backbone were lig...

Embodiment 3

[0124] Example 3, Target Screening for ADCY3 Gene Knockout

[0125] Pig ADCY3 gene information: encodes adenylate cyclase 3 protein; located on pig chromosome 3;

[0126] GeneID is 100514402, Sus scrofa. The protein encoded by the porcine ADCY3 gene is shown in GENBANKACCESSION NO.XP_020943435.1 (linear CON 12-JAN-2018). In genomic DNA, the porcine ADCY3 gene has 22 exons, wherein the third exon is shown in SEQ ID NO: 14, and its encoded protein fragment is shown in SEQ ID NO: 15.

[0127] 1. ADCY3 gene knockout preset target and adjacent genome sequence conservation analysis

[0128] 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).

[0129] 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 12 of pig ADCY3 gene), and then electrophoresis was performed. The PCR amplification products were ...

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PUM

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Abstract

The invention discloses a CRISPR system for ADCY3 gene editing and application of the CRISPR system to construction of obese porcine nuclear transplantation donor cells. The CRISPR/Cas9 system for porcine ADCY3 gene editing comprises a Cas9 expression vector and a gRNA expression vector for a porcine ADCY3 gene, wherein the Cas9 expression vector is a plasmid with a complete sequence as shown in SEQ ID NO.2, the gRNA expression vector expresses gRNA as shown in SEQ ID NO.22, and a target of the gRNA is as shown in SEQ ID NO.18. According to the invention, four gRNAs are designed for the porcine ADCY3 gene, efficient gRNAs are screened from the four gRNAs, then preset targets are knocked out, so that the workload of later monoclonal cell identification and screening can be effectively reduced, and the gene editing efficiency can be detected directly through PCR product sequencing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a CRISPR / Cas9 system for ADCY3 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 la...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10A01K67/027
CPCC12N15/85C07K14/47A01K67/0276C12N2310/20A01K2217/075A01K2227/108A01K2267/0362Y02A50/30
Inventor 牛冬汪滔马翔刘璐曾为俊王磊程锐陶裴裴赵泽英黄彩云
Owner NANJING KGENE GENETIC ENG CO LTD
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