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Inducible DNA methylation editing system based on CRISPR/dCas9

An editing system and methylation technology, applied in the biological field, can solve the problems of off-target and inability to realize time-space specific DNA methylation editing, etc., and achieve the effect of wide application range and flexible use method

Pending Publication Date: 2020-10-09
池嘉栋 +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, methylation modification is a highly dynamic and delicate process, and the above-mentioned methods cannot achieve precise and space-time specific DNA methylation editing. After the CRISPR / Cas9 system enters the cell, it will have a persistent editing effect on the genome, which may cause serious off-target

Method used

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  • Inducible DNA methylation editing system based on CRISPR/dCas9
  • Inducible DNA methylation editing system based on CRISPR/dCas9
  • Inducible DNA methylation editing system based on CRISPR/dCas9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1: light-controlled DNA methylation writing system (CLAMS d ) construction

[0036] Such as figure 1 Shown, CLAMS dConsisting of a three-plasmid system, SpCas9D10A and H840A are mutated to lose endonuclease activity but retain DNA targeting activity, so it is called deadCas9 (dCas9), and the SV40NLS-FLAG-dCas9-CIBN-NLS fusion protein is constructed using the whole gene synthesis method As the anchor element, CRY2PHR was fused to the DNA methyltransferase DNMT3A as the effector element on the other hand. The Cas9 gRNA clone is downstream of the human U6 promoter and expressed by U6. Under dark conditions, gRNA guides the dCas9-CIBN protein to the designated position of the genome, but the DNA methylation effect element is free in the nucleus; under blue light irradiation, CIBN recruits CRY2PHR and carries the activation element to the designated position to edit DNA methylation .

Embodiment 2

[0037] Embodiment 2: light-controlled DNA demethylation system (CLAMS t ) construction

[0038] CLAMS t with CLAMS d The composition is similar, the principle is as figure 2 Shown, CLAMS d It also consists of a three-plasmid system, using the whole gene synthesis method to construct the SV40NLS-FLAG-dCas9-CIBN-NLS fusion protein as the anchor element, and on the other hand, the fusion of CRY2PHR and the demethylase TET1 as the effector element. The Cas9 gRNA clone is downstream of the human U6 promoter and expressed by U6. Under dark conditions, gRNA guides the dCas9-CIBN protein to the designated position of the genome, but the DNA methylation effect element is free in the nucleus; under blue light irradiation, CIBN recruits CRY2PHR and carries the activation element to the designated position, modifying DNA demethylation the process of.

Embodiment 3

[0039] Example 3: Verification of the CLAMS DNA methylation editing system

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Abstract

The invention relates to a DNA methylation editing system based on CRISPR / dCas9 and application of the DNA methylation editing system. Optogenetics is combined with DNA methylation editing, a light-operated DNA methylation editing system based on a CRISPR / dCas9 system is constructed, and accurate space-time regulation and control of DNA methylation editing are realized; the invention provides a CRISPR / dCas9-based inducible DNA methylation editing system, which comprises a guide element, an anchoring element and an editing effect element which can sequentially interact with one another, whereinthe anchoring element comprises a stimuli-responsive protein A and inactivated SpCas9; the editing effect element comprises a stimuli-responsive protein B and a DNA methylation editing effect protein; and the stimuli-responsive protein A and the stimuli-responsive protein B can be mutually combined under the stimulation effect. The invention has the beneficial effects that optogenetics and DNA methylation editing are combined, so that accurate space-time regulation and control of DNA methylation editing are realized; the DNA methylation editing system with blue light response is established under the action of the photosensitive protein, the DNA methylation editing system with different wavelength regulation and control is realized through different photosensitive protein combinations, the use method is flexible, and the application range is wide.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CRISPR / dCas9-based inducible DNA methylation editing system. Background technique [0002] DNA methylation at 5-cytosine (5mC) plays an important role in biological processes, and is of great significance for gene imprinting, cell fate determination, chromatin structure remodeling and gene expression regulation. Changes in DNA methylation modification are closely related to many diseases, such as abnormal embryonic development and tumors. The process of DNA 5mC methylation modification is mainly mediated by DNA methyltransferases, which mainly include DNMT1, DNMT3A and DNMT3B; on the other hand, DNA 5mC modification is a dynamic and reversible process, and the demethylation process is first performed by the TET family Protein-mediated oxidation of 5mC to 5-hydroxymethylated cytosine (5hmC), which is further oxidized to 5-formylcytosine (5fC), which is finally oxidized again to 5-c...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90
CPCC12N15/85C12N15/907C12N2800/107
Inventor 池嘉栋高明郑向前赵杰
Owner 池嘉栋
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