Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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
View PDF3 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for improving homology directed efficiency 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 the Sox18 protein in the 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 the ssDNA template (single-stranded DNA as a repair template), the gene sequence of the Sox18 ssDNA template is shown in SEQ ID NO:6. ssDNA templates can also be purchased from IDT (http: / / idtdna.com). In order to improve the efficiency of precise editing, the 5' and 3' ends of the ssDNA template need to be chemically modified, specifically adding phosphorot...

Embodiment 2

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

[0098] 2.1 Gene editing design and molecular clone

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

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

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

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

[0103] 2.2 Cell culture and transfection: use electrotransfer device: Neon Transfection System;

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

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

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

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

Embodiment 3

[0116] The P2A-EGFP fluorescent protein was accurately 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, design the required single-stranded DNA oligonucleotides according to the sequence, and add 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 The end and the 3' end need to be chemically modified, spec...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com