Method for improving homology directed efficiency in gene editing

A gene editing and homologous recombination technology, applied in the field of gene editing, can solve the problems of reducing the accuracy of gene editing

Active Publication Date: 2020-05-12
WEIFANG MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After a DNA double-strand break occurs, because the NHEJ mechanism runs through the interphase of cell division and is simpler and more effective, cells are more inclined to use

Method used

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  • Method for improving homology directed efficiency in gene editing

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0069] Example 1

[0070] The myc-tag gene was accurately knocked into the C-terminus of Sox18 protein of mouse NIH3T3 cell line by plasmid transfection method

[0071] 1.1 Gene editing design and molecular cloning

[0072] 1.1.1. Use the online CRISPR design tool (http: / / crispor.tefor.net / ) to design the gRNA sequence required for gene editing, and design the required single-stranded DNA oligonucleotides according to the sequence, respectively at the 5'end Add BsbI restriction site;

[0073] The gene sequence of Sox18 gRNA-F is shown in SEQ ID NO: 4;

[0074] The gene sequence of Sox18 gRNA-R is shown in SEQ ID NO: 5;

[0075] 1.1.2 Design ssDNA template (single-stranded DNA as a repair template), the Sox18ssDNA template gene sequence is shown in SEQID NO:6. The ssDNA template can also be purchased from IDT (http: / / idtdna.com). In order to improve the precision editing efficiency, the 5'end and 3'end of the ssDNA template need to be chemically modified, specifically, phosphorothioate...

Example Embodiment

[0096] Example 2

[0097] The myc-tag gene was accurately knocked into the C-terminus of Sox18 protein of mouse NIH3T3 cell line by RNA transfection method

[0098] 2.1 Gene editing design and molecular cologne

[0099] 2.1.1 is the same as 1.1.1 in Example 1;

[0100] 2.1.2 is the same as 1.1.2 in Example 1;

[0101] 2.1.3 is the same as 1.1.3 in Example 1;

[0102] 2.1.4 is the same as 1.1.4 in Example 1;

[0103] 2.2 Cell culture and transfection: use electroporation instrument: Neon Transfection System;

[0104] 2.2.1 is the same as 1.2.1 in Example 1;

[0105] 2.2.2 Prepare a 12-well culture plate, add the culture medium containing 10μM RS-1 and place it in the incubator to preheat;

[0106] 2.2.3 Use pU6-CGA-110 plasmid and MEGAshortscript kit (Ambion, AM1354) to synthesize the target gene gRNA in vitro; (operation steps are standard steps of the kit);

[0107] 2.2.4 Use plasmid pT7-A-120 and Ambion's mMESSAGE mMACHINE Kit (AM1340) to synthesize AUNIP mRNA in vitro; (operation steps are...

Example Embodiment

[0115] Example 3

[0116] Knock into the P2A-EGFP fluorescent protein at the C-terminus of the mouse Hox9A protein to make the expression of Hox9A protein with fluorescent markers.

[0117] 3.1 Gene editing design and molecular cloning

[0118] 3.1.1 Use the online CRISPR design tool (http: / / crispor.tefor.net / ) to design the gRNA sequence required for gene editing, and design the required single-stranded DNA oligonucleotides according to the sequence and add them to the 5'end respectively BsbI restriction site.

[0119] The gene sequence of HoxA9 gRNA-F is shown in SEQ ID NO: 9;

[0120] The gene sequence of HoxA9 gRNA-R is shown in SEQ ID NO: 10;

[0121] 3.1.2 Design ssDNA template,

[0122] The gene sequence of HoxA9 ssDNA template is shown in SEQ ID NO: 11; the ssDNA template can be purchased from IDT (http: / / idtdna.com); it is designed as IDT Ultramer's ssDNA, in order to improve the precision editing efficiency, the 5'of the ssDNA template The end and 3'end need to be chemically m...

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Abstract

The invention relates to a method for improving the homology directed efficiency in gene editing and plasmids used in the method. The method for improving the homology directed efficiency in gene editing comprises the following steps: firstly, conducting target gene editing and design and molecular cloning; and secondly, adding a compound RS-1 into a cell culture medium, conducting culture at 37 DEG C for 24 hours, and then introducing a Cas9 gene segment, an AUNIP gene segment and a target gene gRNA segment into a CRISPR-Cas9 gene editing system by adopting an electroporation transfection method or a micro-injection technology. By means of the method for improving the homology directed efficiency in gene editing, precise gene editing can be conducted on eukaryotic cells, especially mammalian cells, is suitable for any target genes, and includes introduction of point mutation, precise knockout or insertion of small fragments, and the like, and compared with the conventional CRISPR-Cas9system, the method for improving the homology directed efficiency in gene editing has the advantages that the homology directed repair efficiency can be improved by at least 50%, and the homology directed repair efficiency of certain gene loci can even be improved by 2-5 times.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a method for improving the efficiency of homologous recombination in gene editing. Background technique [0002] The new generation of gene editing technology CRISPR has made breakthroughs in many scientific research fields. So far, the genes of various animal and plant cells and stem cells can be systematically edited, which provides the possibility to overcome persistent diseases such as AIDS, various cancers, genetic diseases and rare diseases, and has good research significance. [0003] The most widely used CRISPR system is the CRISPR-Cas9 system. Cas9 is an endonuclease that cuts the paired DNA double strand under the guidance of a guide RNA (gRNA). After the double strand breaks, the cell will start the DNA repair mechanism. To repair broken DNA, since the CRISPR-Cas9 system does not participate in the DNA repair process, in order to carry out precise gene editing, i...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/85C12N15/90C12N15/62C12N15/10
CPCC12N15/79C12N15/85C12N15/902C12N15/102C07K14/4702C12N2800/107C07K2319/41C07K2319/60
Inventor 黄啸王玉冰赵雪
Owner WEIFANG MEDICAL UNIVERSITY
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