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Method for replacing endogenous gene segment in non-homologous end connection manner

An endogenous gene and non-homologous technology, which is applied in the field of replacing endogenous gene fragments by means of non-homologous end joining, can solve the problems of long production cycle and low success efficiency, and achieve the goal of improving production efficiency and high efficiency Effect

Inactive Publication Date: 2020-10-30
南京歆佳医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to overcome the above-mentioned problems of long preparation period and low success rate in gene replacement technology in the existing zebrafish, the purpose of the present invention is to provide a method that does not rely on homologous recombination but relies on non-homologous end joining Efficient method for replacing endogenous gene fragments

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  • Method for replacing endogenous gene segment in non-homologous end connection manner
  • Method for replacing endogenous gene segment in non-homologous end connection manner
  • Method for replacing endogenous gene segment in non-homologous end connection manner

Examples

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Effect test

Embodiment 1

[0061] A method of replacing endogenous gfap gene fragments in a non-homologous end-joining mode of the present invention, comprising the steps of:

[0062] (1) Take the intron and UTR of the untranslated segment of the gfap gene in the genome as the target segment, and determine a guide RNA target sequence (two in total) on both sides of the target segment; determine the intron in the genome for the target segment A left homology arm sequence and a right homology arm sequence, each of which includes a respective guide RNA target sequence;

[0063] (2) Prepare a replacement donor plasmid or a donor DNA fragment, which includes the following sequentially connected elements: left homology arm sequence, middle exogenous replacement sequence, and right homology arm sequence;

[0064] (3) co-introduce the donor plasmid or donor DNA fragment, 2 guide RNAs or DNA that can form the 2 guide RNAs, Cas9 protein or mRNA or DNA that can form Cas9 protein into the target cell to obtain exog...

Embodiment 2

[0078] A method of replacing endogenous th gene fragments in a non-homologous end joining method of the present invention comprises the following steps:

[0079] (1) Take the intron of the untranslated segment and the exon of the translated segment of the target gene in the genome as the target segment, and determine a guide RNA target sequence (two in total) on both sides of the target segment; The segment determines the left homology arm sequence and the right homology arm sequence in the genome, and both the left homology arm sequence and the right homology arm sequence comprise respective guide RNA target sequences;

[0080] (2) Prepare a replacement donor plasmid or a donor DNA fragment, which includes the following sequentially connected elements: left homology arm sequence, middle exogenous replacement sequence, and right homology arm sequence;

[0081] (3) co-introduce the donor plasmid or donor DNA fragment, 2 guide RNAs or DNA that can form the 2 guide RNAs, Cas9 pro...

Embodiment 3

[0096] A method of replacing endogenous th gene fragments in a non-homologous end joining method of the present invention comprises the following steps:

[0097] (1) Use the intron of the untranslated segment of the target gene in the genome as the target segment, and determine a guide RNA target sequence (two in total) on both sides of the target segment; determine the left homonym in the genome for the target segment The source arm sequence and the right homology arm sequence, the left homology arm sequence and the right homology arm sequence all comprise respective guide RNA target sequences;

[0098] (2) Prepare a replacement donor plasmid or a donor DNA fragment, which includes the following sequentially connected elements: left homology arm sequence, middle exogenous replacement sequence, and right homology arm sequence;

[0099] (3) co-introduce the donor plasmid or donor DNA fragment, 2 guide RNAs or DNA that can form the 2 guide RNAs, Cas9 protein or mRNA or DNA that ...

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Abstract

The invention discloses a method for replacing endogenous gene segments in a non-homologous end connection manner so as to realize various complex gene editing. Cas9 nuclease is utilized to simultaneously generate two double-stranded fractures in a genome and donor DNA, and replacement of DNA fragments between two double-stranded fractures in the genome by fragments between two double-stranded fractures of the donor DNA is realized so that a new way is provided for efficiently realizing complex targeted gene editing.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a genome-modifying construct and a transforming method. Background technique [0002] Zebrafish is one of the important model animals for life science research. Compared with humans, the tissues and organs of zebrafish are highly conserved and representative. At the same time, the zebrafish is small in size and its juveniles are transparent. Under the current laboratory technical conditions, it is convenient to dynamically observe the development of tissues and organs and various physiological activities. In order to give full play to the many advantages of zebrafish in research, it is necessary to establish a variety of manipulation methods for its genes, to prepare mutants with loss of gene function or to introduce foreign genes into specific gene loci. Currently, methods for generating gene loss of function in zebrafish are well established and widely used. However, the trans...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/65C12N15/90A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/40A01K2267/03C12N9/22C12N15/113C12N15/65C12N15/902C12N2310/20
Inventor 顾珊烨
Owner 南京歆佳医药科技有限公司
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