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CRISPR (clustered regularly interspaced short palindromic repeats) system for double-target gene editing and application of CRISPR system in construction of depression porcine nuclear transplantation donor cells

A gene editing and gene technology, applied in genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve the huge difference in mouse body size, organ size, physiology and pathology, and cannot truly simulate human physiology. , pathological state and other problems, to achieve the effect of high cost, high difficulty and high feeding cost

Active Publication Date: 2021-11-02
NANJING KGENE GENETIC ENG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 (clustered regularly interspaced short palindromic repeats) system for double-target gene editing and application of CRISPR system in construction of depression porcine nuclear transplantation donor cells
  • CRISPR (clustered regularly interspaced short palindromic repeats) system for double-target gene editing and application of CRISPR system in construction of depression porcine nuclear transplantation donor cells
  • CRISPR (clustered regularly interspaced short palindromic repeats) system for double-target gene editing and application of CRISPR system in construction of depression porcine nuclear transplantation donor cells

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 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).

[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

[0104] Embodiment 2, the effect comparison of plasmid pX330 and plasmid pKG-GE3

[0105] Select high-efficiency gRNA targets located at the RAG1 gene:

[0106] Target of RAG1-gRNA4: 5'-AGTTATGGCAGAACTCAGTG-3' (SEQ ID NO.9).

[0107] Primers used to amplify and detect target-containing fragments are as follows:

[0108] RAG1-nF126: 5'-CCCCATCCAAAGTTTTTAAAGGA-3' (SEQ ID NO. 10);

[0109] RAG1-nR525: 5'-TGTGGCAGATGTCACAGTTTAGG-3' (SEQ ID NO.11)

[0110] Primary porcine fibroblasts were prepared from the ear tissue of newborn Congjiang pigs (female, blood type AO).

[0111] 1. Preparation of recombinant plasmids

[0112] 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 ba...

Embodiment 3

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

[0135] Pig BDNF gene information: encodes brain derived neurotrophic factor protein; located on pig chromosome 2; GeneID is 397495, Sus scrofa. The protein encoded by the porcine BDNF gene is shown in GENBANK ACCESSIONNO.XP_005654741.1 (linear CON 12-JAN-2018). In the genomic DNA, the porcine BDNF gene has 6 exons, wherein the 6th 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.

[0136] 1. BDNF gene knockout predetermined target and conservation analysis of adjacent genome sequences

[0137] 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).

[0138] Genomic DNA of 18 pigs was respectively used as templates, and primer pairs (the target sequence of the primer pairs included exon 6 of porcine BDNF gene) were used for PCR amplification, and then ele...

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PUM

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Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) system for double-target gene editing and application of the CRISPR / Cas9 system in construction of depression pig nuclear transplantation donor. The CRISPR / Cas9 system for pig BDNF and SLC6A4 gene editing. The CRISPR / Cas9 system comprises a Cas9 expression vector, a gRNA expression vector aiming at a pig BDNF gene and a gRNA expression vector aiming at a pig SLC6A4 gene, and the complete plasmid sequence of the Cas9 expression vector is as shown in SEQ ID NO. 2. When the screened gRNA combined modified Cas9 efficient expression vector is used for gene editing, the editing efficiency is remarkably improved compared with that of the original vector.

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 BDNF and SLC6A4 and an application thereof. Background technique [0002] Depression is the most common depressive disorder, with significant and persistent low mood as the main clinical feature, and it is the main type of mood disorders. At present, there are more than 264 million depressed patients in the world, and depression has become the second largest disease in the world. In my country, the number of patients with depression has reached 90 million, but the medical prevention and treatment of depression in our country is still in a situation of low recognition rate, and the incidence of depression has begun to show a trend of younger age. [0003] So far, the etiology of depression is not very clear, but it is certain that many factors such as genetics, psychology and social environment are involved in the pathogenesis of d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/55C12N15/12C12N5/10
CPCC12N15/85C12N9/22C07K14/475C07K14/47C12N5/0656C12N2800/107C12N2510/00Y02A50/30
Inventor 牛冬汪滔马翔曾为俊刘璐王磊程锐赵泽英陶裴裴黄彩云
Owner NANJING KGENE GENETIC ENG CO LTD
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