A method to improve the efficiency of homologous recombination in gene editing

A technology of gene editing and homologous recombination, applied in the field of gene editing, can solve the problems of reducing the accuracy of gene editing, achieve the effect of improving the efficiency of homologous recombination and promoting the repair mechanism

Active Publication Date: 2022-03-25
WEIFANG MEDICAL UNIV
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  • Description
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  • Application Information

AI Technical Summary

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 the NHEJ mechanism to repair damaged DNA; this leads to the introduction of various random mutations due to NHEJ in gene editing , thereby reducing the precision of gene editing

Method used

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  • A method to improve the efficiency of homologous recombination in gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Precise knock-in of the myc-tag gene at the C-terminus of Sox18 protein in mouse NIH3T3 cell line by plasmid transfection

[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 a ssDNA template (single-stranded DNA as a repair template), the Sox18 ssDNA template gene sequence is shown in SEQ ID NO: 6. ssDNA templates can also be purchased from IDT (http: / / idtdna.com). In order to improve the precise editing efficiency, chemical modification is required at the 5' and 3' ends of the ssDNA template, specifically by adding phosphorothioate bon...

Embodiment 2

[0097] Precise knock-in of myc-tag gene at the C-terminus of Sox18 protein in mouse NIH3T3 cell line by RNA transfection

[0098] 2.1 Gene editing design and molecular clones

[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 electroporator: 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 culture medium containing 10 μM RS-1, and place it in an incubator to preheat;

[0106] 2.2.3 Use pU6-CGA-110 plasmid and MEGAshortscript kit (Ambion, AM1354) to synthesize 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 standard steps of the kit);

[...

Embodiment 3

[0116] The P2A-EGFP fluorescent protein was precisely knocked into the C-terminus of the mouse Hox9A protein, so that the expression of the Hox9A protein was fluorescently labeled.

[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 the HoxA9 ssDNA template is shown in SEQ ID NO: 11; the ssDNA template can be purchased from IDT (http: / / idtdna.com); the ssDNA designed as IDT Ultramer, in order to improve the precision editing efficiency, the 5' of the ssDNA template Chemical modifications a...

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Abstract

A method for improving homologous recombination efficiency in gene editing and a plasmid used in the method, comprising the following steps: first, carrying out target gene editing design and molecular cloning; secondly, adding compound RS-1 to a cell culture medium, and Cultivate at 37°C for 24 hours, then introduce Cas9 gene fragments, AUNIP gene fragments, and target gene gRNA fragments into the CRISPR-Cas9 gene editing system by electroporation transfection or microinjection technology; the method of the present invention improves homology in gene editing The method of recombination efficiency can carry out precise gene editing on eukaryotic cells, especially mammalian cells, and is suitable for any target gene, such as introducing point mutations, precise knockouts or inserting small fragments, etc. Compared with the conventional CRISPR‑Cas9 system, it can improve The homologous recombination repair efficiency is at least 50%, and it can even achieve a 2-5 times improvement for certain gene loci.

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] CRISPR, a new generation of gene editing technology, 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 intractable 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 off its paired DNA double-strand under the guidance of a guide RNA (gRNA). After the double-strand break, the cell will initiate a DNA repair mechanism. In order to repair the broken DNA, since the CRISPR-Cas9 system does not participate in the DNA repair process, in order to carry out preci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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 UNIV
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