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CRISPR/CasRx-based gene editing method and application thereof

A construction method and targeted technology, applied in the field of genetic engineering, can solve problems such as lack of treatment for hereditary deafness, improve the morphology of hair cells and ciliary bundles, have no off-target effects, and prevent progressive hearing loss.

Active Publication Date: 2021-11-05
EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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Problems solved by technology

[0007]There is still a lack of research on the use of CRISPR-Cas13 RNA editing system to treat hereditary deafness

Method used

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  • CRISPR/CasRx-based gene editing method and application thereof
  • CRISPR/CasRx-based gene editing method and application thereof
  • CRISPR/CasRx-based gene editing method and application thereof

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Embodiment Construction

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the examples. It must be pointed out that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the present invention . Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.

[0034] 1. Examples of implementation methods

[0035] (1) CasRx specific knockout of Tmc1 in vitro Bth transcript

[0036] To knock down Tmc1 efficiently and specifically in HEK293T cells Bth mRNA, first compared two RNA editing systems, PspCas13b and CasRx, both of which were shown to efficiently knock down endogenous transcripts. Then 30 Tmc1-targeted Bth The sgRNAs of the transcript, in the PspCas13b and CasRx systems, th...

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Abstract

The invention provides sgRNA of a targeted Tmc1 mutant (c.1235T > A; p.M412K), an expression vector, a CRISPRs-CasRx system, a kit and application of the sgRNA, and belongs to the technical field of genetic engineering. The sequence of sgRNA of the targeted Tmc1 mutant is shown as SEQIDNO.3; and CasRx containing the sgRNA can enable a Tmc1Bth transcript to be reduced by 82% under the condition that wild Tmc1 + is not interfered, CasRx based on an AAV-PHP.eB carrier is injected into inner ears of newborn Beethoven mice, it is found that Tmc1Bth is reduced by 70% within two weeks, and miss-target is not detected in a whole transcriptome. The sgRNA targeted Tmc1 mutant provided by the invention is high in specificity, and can improve the survival rate of Tmc1Bth mutant mouse hair cells, so that the form of cilia bundles is recovered, the mechanical transduction current is reduced, and progressive hearing loss is remarkably improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene editing method based on CRISPR-CasRx and an application thereof. Background technique [0002] According to the World Health Organization (WHO), hearing loss is one of the most common sensory deficits, affecting approximately 5% of the world's population, 34 million of whom are children. In children, hearing loss affects cognitive, language, and psychosocial development. Almost half of deafness cases are caused by genetic factors. Among different types of hereditary hearing loss, 20-25% of non-syndromic hearing loss ( NSHL) cases are autosomal dominant. So far, more than 100 genes have been confirmed to be related to NSHL, and autosomal dominant inheritance is mostly progressive hearing loss, so there is enough time window for gene therapy. [0003] TMC1 (transmembrane channel class 1) is the sixth most common inherited deafness gene, and mutation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N15/65C12N15/55A61K48/00A61P27/16
CPCC12N15/113C12N15/86C12N15/65C12N9/22A61K48/005A61P27/16C12N2750/14143C12N2800/22C12N2310/20
Inventor 舒易来胡晓湘李耕林郑子文李果崔冲
Owner EYE & ENT HOSPITAL SHANGHAI MEDICAL SCHOOL FUDAN UNIV
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