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A kind of sgRNA guide sequence and application of specific targeting human abcb1 gene

A kind of specific, gene technology, applied in sgRNA guide sequence and application field, to achieve the effect of solving the problem of multi-drug resistance

Active Publication Date: 2019-03-22
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CRISPR-Cas9 technology also has the disadvantage of context dependence, and currently it can only be applied to target sites with upstream PAM sequences

Method used

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  • A kind of sgRNA guide sequence and application of specific targeting human abcb1 gene
  • A kind of sgRNA guide sequence and application of specific targeting human abcb1 gene
  • A kind of sgRNA guide sequence and application of specific targeting human abcb1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) sgRNA design

[0038] According to the genome sequence of human ABCB1 gene (gene ID: 5343), two sgRNAs targeting human ABCB1 gene were designed. The 20nt oligonucleotide sgRNA guide sequence is: Sg1: 5'-TTGGACTGTCAGCTGCTGTC-3' (located in the eighth exon of gene ABCB1) and Sg2: 5'-TGGACTTCCTCTCATGATGC-3' (located in the fifth exon of gene ABCB1 exon)( figure 1 ); add CACCG to its 5' end to obtain the forward oligonucleotide (Forward oligo); obtain its corresponding DNA complementary strand according to the guide sequence, and add AAAC to its 5' end to obtain the reverse oligonucleotide ( Reverse oligo). Synthesize the above-mentioned forward oligonucleotides and reverse oligonucleotides respectively, denature the Forward oligo and Reverse oligo of the synthesized sgRNA oligonucleotides in pairs, and anneal; At the same time, the designed gRNA target sequence was blasted to exclude non-specific target cutting sites. The specific oligonucleotide sequence is shown i...

Embodiment 2

[0046] (1) Co-transfection of core plasmid and packaging plasmid pMD2.G and psPAX2 into 293T cells

[0047] Cultivate 293T cells until the confluence rate of 293T cells reaches 50% to 60%, 12 to 18 hours after seeding is the best time for transfection; replace fresh culture medium before transfection, add 3mL medium to a 60mm dish; The amount used is 4 μg of core plasmid (lentiCRISPRv2-hABCB1-Sg1 or lentiCRISPRv2-hABCB1-Sg 2 prepared in Example 1, and lentiCRISPRv2 empty vector as a control), 3 μg of psPAX2, 1 μg of pMD2.G, 24 μL of PEI, supplemented with DMEM to The total volume is 200 μL, and the order of addition is DMEM, PEI, and plasmid DNA (core plasmid, pMD2.G, and psPAX2); then stand at room temperature for 30 minutes to fully polymerize PEI and plasmid DNA. After polymerization, add the transfection system dropwise to the above Culture a small dish with 293T cells, shake it gently and place it at 37°C with 5% CO 2 Continue culturing in the incubator;

[0048] (2) Vi...

Embodiment 3

[0061] Example 3 In vitro cell experiments to identify the knockout effect of the human gene ABCB1 on the cells after screening

[0062] (1) western blot

[0063] The p-gp protein expression levels of the cell lines HCT-8 / Vsg1, HCT-8 / V sg2, KBV200sg1 and KBV200sg2 successfully knocked out of the ABCB1 gene in Example 2 were identified by western blot experiments, and the cell lines successfully transfected with the empty vector lentiCRISPRv2 HCT-8 / V and KBv200 were used as controls, wherein the primary antibody was Anti-ABCB1 (MM SC-13131, purchased from Santa Cruze), the secondary antibody was Anti-mouse IgG, HRP-linked Antibody (product number 7076, cellsignaling), and the specific The method is a conventional western blot operation procedure.

[0064] The result is as Figure 4 As shown, in HCT-8 / V and KBv200 cells, there is a significant difference in the expression of p-gp between the cells transfected with sg1 and sg2 and the cells only transfected with the vector, and...

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Abstract

The invention belongs to the field of gene engineering applications and particularly relates to a sgRNA targeting sequence of a specific target human ABCB1 gene and an application. The sgRNA targeting sequence of the specific target human ABCB1 gene is Sg1 or Sg2; the nucleotide sequences of Sg1 and Sg2 are as follows: Sg1: 5'-TTGGACTGTCAGCTGCTGTC-3'; Sg2: 5'-GACTGTCAGCTGCTGTCGTC-3'. Two single-stranded oligo sequences are designed and synthesized according to the sgRNA targeting sequence, double strands are formed through annealing and then connected with a Cas9 carrier, sgRNA and a CRISPR system are introduced into a target cell through the Cas9 carrier, Cas9 protein can find a matched DNA sequence under the guidance of sgRNA, cutting is performed, and the ABCB1 gene is knocked out.

Description

technical field [0001] The invention belongs to the field of genetic engineering applications, in particular to a sgRNA guide sequence specifically targeting human ABCB1 gene and its application. Background technique [0002] CRISPR-Cas9 is an immune invasion system evolved during the evolution of bacteria and archaea to resist foreign invasion, including defense against invading viruses and foreign DNA. In the field of modern genetic engineering applications, it has become the three major genome editing tools alongside TALEN (transcription activator-like effector nuclease) and ZFN (zinc-finger nuclease) technologies. Compared with TALEN and ZFN technology, CRISPR-Cas9 technology has specific DNA recognition ability. In the second type of CRISPR system, Cas9 endonuclease cuts double-stranded DNA under the guidance of sgRNA, causing double-strand breaks in the genome. Repair instability produces non-specific recombination to generate repair errors (insertion or deletion), wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867A61K31/7088A61P35/00
CPCC07K14/47C12N15/113C12N15/86C12N2740/15043C12N2800/107C12N2800/80
Inventor 石智杨阳邱建阁张文姬蒋起韦覃武明陈耀郑迪威魏梦宁
Owner JINAN UNIVERSITY
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