Mitochondrial DNA editing system based on TALE assembly

An editing system and mitochondrial technology, applied in the field of gene editing, can solve the problems that DNA editing cannot be used and cannot be used to construct mitochondrial DNA mutant animal models, etc.

Active Publication Date: 2021-09-17
NANJING MEDICAL UNIV
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Problems solved by technology

Using ZFN and TALEN plus mitochondrial localization signal (MTS), the mutant mitochondrial DNA can be cut, causing the mitochondrial DNA with double-strand breaks to be degraded, but this method cannot be used to construct mit

Method used

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  • Mitochondrial DNA editing system based on TALE assembly
  • Mitochondrial DNA editing system based on TALE assembly
  • Mitochondrial DNA editing system based on TALE assembly

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Experimental program
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Embodiment 1

[0061] Using the mitochondrial DNA editing system based on high-efficiency assembly TALE, the zebrafish mitochondrial DNA mutation model was constructed and the human disease model was simulated.

[0062] (1) Selection of RVD module and assembly of TALE

[0063] The pathogenic mutation sites of C / T to G / A of G3733 and G8363 on human mitochondrial DNA (reference sequence version: NC_012920.1) were selected, and the corresponding target sites were screened according to conservation analysis ( Figure 4 and Figure 5 ). For these two pathogenic mutation loci, the corresponding loci G4247 and G8892 on zebrafish were designed (reference sequence version: NC_002333.2). According to the DNA sequence, the RVD at the corresponding position is selected in the RVD library of the present invention, for example: the first and second bases of the DNA sequence are GT, and the RVD module that recognizes the GT sequence at the first position in the RVD library is selected; when the third and...

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Abstract

The invention belongs to the technical field of gene editing, and discloses a mitochondrial DNA editing system based on TALE assembly. The editing system comprises an RVD library, a mitochondrial localization skeleton carrier and a cell nucleus localization skeleton carrier, and the RVD library comprises at least 192 RVD modules including 160 double RVD modules, 16 single RVD modules and 16 half RVD modules. According to the editing system, the assembly efficiency of a TALE module can be remarkably improved, and the assembly cost and time of the TALE module can be remarkably reduced. According to the mitochondrial DNA editing system based on the TALE assembly, the assembly method is easy to operate and suitable for application and popularization. By means of the method, mitochondrial DNA editing can be rapidly and efficiently achieved, and the method is used for manufacturing various animal models so as to simulate diseases caused by human mitochondrial DNA mutation; the method can be potentially applied to gene therapy of mitochondrial DNA mutation diseases.

Description

technical field [0001] The invention belongs to the technical field of gene editing, in particular to a mitochondrial DNA editing system based on TALE assembly. Background technique [0002] Transcription activator-like effector (TALE) is the core module of second-generation gene editing tools, which is mainly composed of several 34 amino acid repeating units, and uses two repeating variable residues (RVD, repeat-variable di-residue) specifically recognizes DNA sequences. A variety of methods have been reported for the assembly of TALE modules, but most of these methods are tedious, time-consuming, laborious, and expensive, so how to efficiently assemble customized TALEs has become a difficulty in its promotion and application. [0003] Mitochondrial diseases are a class of major diseases characterized by abnormal mitochondrial function. Mitochondrial diseases are often caused by nuclear gene mutations and mitochondrial DNA mutations, and mitochondrial DNA mutations usuall...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027C40B40/08C40B40/10
CPCC12N15/8509A01K67/0275C40B40/10C40B40/08C12N2800/106A01K2227/40A01K2267/03
Inventor 沈彬王建瑛戴奕晨陈晓旭郭佳银
Owner NANJING MEDICAL UNIV
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