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PspCas13b-Alkbh5 single gene specific m6A modification editing method

A specific and specific technology applied in the field of specific editing of single-gene mRNAm6A modification

Pending Publication Date: 2019-07-26
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no specific demethylation method for target gene mRNA

Method used

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  • PspCas13b-Alkbh5 single gene specific m6A modification editing method
  • PspCas13b-Alkbh5 single gene specific m6A modification editing method
  • PspCas13b-Alkbh5 single gene specific m6A modification editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 The PspCas13b-Alkbh5 / gRNA system achieves CYB5A mRNA demethylation and regulates its expression level.

[0034] 1 Experimental materials and methods

[0035] 1.1 Main reagents and instruments

[0036] The HEK293T cell line was kept in our laboratory or sourced from other routine laboratory laboratories and cell banks. Fetal bovine serum was purchased from Gbico Company, cell transfection reagent, siRNA blank control, and si-GPR30 were purchased from Ribo Biotechnology Co., Ltd., G-1 and other reagents were from Sigma Company (analytical grade or above). The Cell Counting Kit-8 kit was purchased from Tongren Chemical Company, the fluorescence microscope was purchased from Olympus Company, and the fluorescence quantitative PCR instrument was purchased from Olympus Company 480II was purchased from Roche Company, and laser confocal microscope LSM710 was purchased from Zeiss Company.

[0037] 1.2 Cell Recovery and Culture

[0038] The HEK293T cell line frozen ...

Embodiment 2

[0049] Example 2 PspCas13b-Alkbh5 / gRNA system explores CTNNB1 5'UTR m 6 Effect of A modification on mRNA expression

[0050] 1 Experimental materials and methods

[0051] 1.1 Western-Blotting analysis

[0052] The treated cells were collected, washed twice with PBS, added with three decontaminated cell lysates, and placed on ice for 15-20min. Collect the cell lysate in EP tube, centrifuge at the maximum speed for 20min, and collect the supernatant. Protein concentration was determined by Bradford method. SDS-PAGE electrophoresis separation was carried out at a voltage of 80-120 V with 20 μg protein loading per well. Under the condition of constant current 200mA, 90min, electrotransfer to methanol pretreated PVDF membrane. 5% skimmed milk powder (prepared in PBST) blocked the PVDF membrane for 2h. The detection antibody was diluted with blocking solution at a ratio of 1:1000, and the PVDF membrane was incubated overnight at 4°C. Wash the PVDF membrane with PBST for 10 mi...

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Abstract

The invention discloses application of demethylation modification of targeted mRNA by using PspCas13b in combination with m6A demethylase Alkbh5. A study shows that a PspCas13b-Alkbh5 fusion protein can perform demethylation modification by targeting target gene (such as CYB5A and CTNNB1) mRNA through the specific gRNA to further regulate the target gene expression. m6A RNA immunoprecipitation assays and m6A site quantitative PCR results show that the PspCas13b-Alkbh5 fusion protein can effectively reduce the m6A methylation level of the target gene mRNA in the cell, and no off-target phenomenon is found. The PspCas13b is used as a means and combined with the m6A demethylase Alkbh5 to achieve the demethylation modification of the targeted mRNA for the first time and regulate the expressionof the specific purpose and application accordingly. The method breaks through the previous specific protein expression regulation based on previous change of DNA genetic information, provides a newpotential treatment mode and strategy for treatment of various diseases in the future, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of modification and editing of single gene mRNA, in particular, the invention relates to a method for specifically editing single gene mRNA m 6 A modified method. Background technique [0002] The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is a unique acquired immune system in some bacteria and archaea. This system is guided by a specific sequence of RNA to specifically cut and degrade exogenous DNA. The CRISPR / Cas system can be divided into three types, among which the type II CRISPR / Cas system has been transformed into a tool for targeted genome editing due to its simple composition. By artificially designing and transcribing RNA in vitro, sgRNA (single guide RNA) with a guiding effect can be synthesized, and the sgRNA guides the Cas protein to specifically cut the target DNA sequence. Through the modification of the Cas protein, under the guidance of sgRNA, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90
CPCC12N15/907
Inventor 王红胜黎婕昕
Owner SUN YAT SEN UNIV
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