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SgRNA directing sequence for specific targeting of human ABCG2 gene and application thereof

A kind of specific and genetic technology, applied in the field of sgRNA guide sequence specifically targeting human ABCG2 gene

Active Publication Date: 2016-12-07
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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  • SgRNA directing sequence for specific targeting of human ABCG2 gene and application thereof
  • SgRNA directing sequence for specific targeting of human ABCG2 gene and application thereof
  • SgRNA directing sequence for specific targeting of human ABCG2 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) sgRNA design

[0038] According to the genome sequence of human ABCG2 gene (gene ID: 9429), a sgRNA targeting human ABCG2 gene was designed. The 20nt oligonucleotide sgRNA guide sequence is: Sg1: 5'-GCTGCAAGGAAAGATCCAAG-3' (located in the third exon of gene ABCG2) ( figure 1 ); add CACCG to its 5' end to obtain a forward oligonucleotide (Forward oligo) (see Sg1-F in Table 1); obtain its corresponding DNA complementary strand according to the guide sequence, and add Add C to AAAC and its 3' end to obtain a reverse oligonucleotide (Reverse oligo) (see Sg1-R in Table 1). 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 in Table 1.

[0039] Table 1 Oligonuc...

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-hABCG2-Sg1 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 a total volume of 200 μL, and the order of addition DMEM, PEI, and plasmid DNA (core plasmid, pMD2.G, and psPAX2) respectively; then stand at room temperature for 30 minutes to fully polymerize PEI and plasmid DNA, and add the transfection system dropwise to the above-mentioned small dish with 293T cells after polymerization , shake gently and place in 37°C, 5% CO 2 Continue culturing in the incubator;

[0048] (2) Virus harvest and concentrati...

Embodiment 3

[0061] Example 3 In vitro cell experiments to identify the knockout effect of the human gene ABCG2 on the cells after screening

[0062] (1) western blot

[0063] The ABCG2 protein expression level of the cell line S1M1-80sg1, which successfully knocked out the ABCG2 gene in Example 2, was identified by western blot experiments. The cell line S1M1-80 successfully transfected with the empty vector lentiCRISPRv2 was used as a blank control, S1 was used as a positive control, and S1M1-80 was used as a positive control. 80 as a negative control; among them, the primary antibody is Anti-ABCG2 (MM SC-58222, purchased from santacruze), the secondary antibody is Anti-mouse IgG, HRP-linked Antibody (product number 7076, cell signaling), and the specific method is conventional western blot operation process.

[0064] The result is as Figure 4 As shown, in S1M1-80 cells, there is a significant difference in the expression of ABCG2 between the cells transformed into sg1 and the cells o...

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Abstract

The invention discloses an sgRNA directing sequence for specific targeting of a human ABCG2 gene and an application thereof, and belongs to the field of gene engineering application. The sgRNA directing sequence has a nucleotide sequence of GCTGCAAGGAAAGATCCAAG. According to the sgRNA directing sequence, two single-stranded oligo sequences are designed and synthesized and then are annealed to form a double chain, and then the double chain is connected with a Cas9 vector; with use of the Cas9 vector, sgRNA and a CRISPR system are introduced into target cells, a Cas9 protein can find out a DNA sequence matched with the Cas9 protein under guidance of the sgRNA, shearing is performed, and the ABCG2 gene is knocked out. According to the sgRNA directing sequence provided by the invention, the ABCG2 gene is knocked out or edited by the CRISPR-Cas9 system, then the expression of the ABCG2 is inhibited or eliminated, and the problem of multi-drug resistance generated in treatment of tumor can be effectively solved.

Description

technical field [0001] The invention belongs to the field of genetic engineering applications, and in particular relates to a sgRNA guide sequence specifically targeting human ABCG2 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 d...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/86C12N5/10A61K48/00A61P35/00
CPCA61K48/00C07K14/47C12N15/113C12N15/85C12N2800/107
Inventor 石智陈耀张文姬魏梦宁邱建阁蒋起韦杨阳郑迪威王昆黄家荣
Owner JINAN UNIVERSITY
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