RNA editing system based on CRISPR-Cas13 and application of RNA editing system

An RNA editing and editing technology, applied in the field of CRISPR-Cas13-based RNA editing system and gene editing, which can solve the problems of precise regulation without chemical modification and the inability to achieve precise time and space control of RNA editing.

Active Publication Date: 2020-05-29
TIANJIN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, none of these systems can achieve precise spatiotemporal control of RNA editing, which is critical for understanding the dynamic function of RNA and its relationship with cellular phenotype
Although RNA modification has an important impact on its function, there is no effective method to precisely regulate the chemical modification of specific genes

Method used

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  • RNA editing system based on CRISPR-Cas13 and application of RNA editing system
  • RNA editing system based on CRISPR-Cas13 and application of RNA editing system
  • RNA editing system based on CRISPR-Cas13 and application of RNA editing system

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Experimental program
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Effect test

Embodiment

[0059] Plasmid construction

[0060] Synthesized human codon-optimized CIBN and dCas13b (R146A / H151A / K385A / R394A) and cloned their fusion protein into pcDNA3.1 + The vector ensures that the open reading frame has no frame shift and mutation. Synthesize the fusion protein of human Mettl3 methylase domain (369-580 amino acids) and Mettl14 methylase domain (116-402 amino acids), and clone the fusion protein into pCRY2PHR-mCherryN1 (Addgene 26866), the restriction sites are SmaI and XbaI, which are the response elements of PAMEC-2; the full-length sequence of human FTO was synthesized and cloned into pCRY2PHR-mCherryN1 using the same strategy as above, which is the response element of PAMEC-1. The guide RNA was cloned into the Cas13bcrRNA backbone plasmid (Addgene 103853), and the restriction site was BbsI.

[0061] cell transfection

[0062] The present invention verified the effectiveness of the PAMEC system in Hela cells and mouse bone marrow mesenchymal stem cells. Hela cel...

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Abstract

The invention provides an RNA editing system based on CRISPR-Cas13 and application of the RNA editing system. The RNA editing system comprises a targeting element, an editing effect element and a guide element, wherein the targeting element comprises a stimulus-responsive protein A and an inactivated Cas13 enzyme (dCas13), and the editing effect element comprises a stimulus-responsive protein B and an RNA editing effect protein; the stimuli-responsive protein A and the stimuli-responsive protein B are combined with each other under the action of stimuli and are uncombined after the stimuli disappear; the concept of light-operated RNA editing is provided for the first time, optogenetics and RNA editing are combined, the light-operated RNA editing system based on the CRISPR-Cas system is constructed, accurate space-time regulation and control of RNA editing are achieved, and the system has wide application prospects and huge market value.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a gene editing method, in particular to a CRISPR-Cas13-based RNA editing system and its application. Background technique [0002] Ribonucleic acid (RNA) is a genetic information carrier that exists in biological cells and some viruses and viroids. It plays a vital role in the life process. Its main function is to realize the expression of genetic information on proteins. A bridge in the process of transforming genetic information into phenotypes. Epitranscriptomics (Epitranscriptomics) is one of the recently emerging hot fields, which mainly studies the impact of chemical modifications carried by RNA on gene expression. To date, more than one hundred chemical modifications have been discovered on RNA. These modifications are widely distributed on non-coding RNAs, especially rRNA, tRNA and snRNA, and are necessary for the normal function of ncRNA in translation and splicing. Among th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/87C12N15/85C12N5/10A61K48/00A61K38/46A61K38/45A61K38/44A61P19/00A61P19/10A61P19/08A61P25/28A61P25/00A61P9/12A61P9/10A61P3/10
CPCC12N15/87C12N15/85C12N5/0663C12N5/0693C12N5/0682A61K48/005A61K38/465A61K38/45A61K38/44A61P19/00A61P19/10A61P19/08A61P25/28A61P25/00A61P9/12A61P9/10A61P3/10C12N2800/107C12N2510/00
Inventor 马信龙赵杰
Owner TIANJIN HOSPITAL
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