A method for reducing concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing and its application

A technology of gene editing and fragmentation, which is applied in the field of genetic engineering, can solve problems affecting technical accuracy, methods of reducing concatenation, gene targeting failure, etc., and achieve the effect of eliminating DNA concatenation, reducing concatenation, and reducing concatenation rate

Active Publication Date: 2020-11-06
GEMPHARMATECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In most cases, traditional PCR analysis failed to identify these multiple integration events, which seriously affected the precision of the technique (see Skryabin BV, et al. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR -Cas9-mediated genome editing events. Sci Adv. 2020;6(7):eaax2941.)
[0006] In the process of CRISPR-Cas9 gene targeting, there is a head-to-tail tandem phenomenon of recombinant DNA fragments, which leads to the failure of gene targeting
In the field, Southern, qPCR, PCR and other methods are usually used to detect whether there is tandem, but there is no effective method to reduce tandem

Method used

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  • A method for reducing concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing and its application
  • A method for reducing concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing and its application
  • A method for reducing concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] In this example, a method for reducing the concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing is provided.

[0052] (1) Construction of dsDNA

[0053] The schematic diagram of the structure of the dsDNA constructed in this example is as follows figure 1 shown;

[0054] Put DTA forward at the 5' end of dsDNA, CMV forward at the 3' end of dsDNA, and both ends of the dsDNA fragment contain EcoRV restriction sites;

[0055] Construct the designed sequence on the plasmid (Plasmid) vector, and use EcoRV to digest and purify to obtain dsDNA;

[0056] Among them, the enzyme digestion system is EcoRV 3 μL, plasmid 25 μL (concentration is 2 μg / μL), Buffer smart 10 μL, ddH 2 Make up to 100 μL with O;

[0057] Enzyme digestion process: time 8 h; temperature 37°C;

[0058] After digestion, purify and recover, and dilute the dsDNA to 5 ng / uL with TE buffer.

[0059] (2) Microinjection of mouse fertilized eggs

[0060] Obtain fertilized eggs, prepare fix...

Embodiment 2

[0074] In this example, DTA is placed forward at the 5' end of dsDNA, and CMV is placed forward at the 3' end of dsDNA. At the same time, both ends of the dsDNA fragment contain restriction sites for BamH1 and Sac1, and the designed sequence is constructed on the plasmid The vector was digested with BamH1 and Sac1 and purified to obtain dsDNA.

[0075] The digestion system is: BamH1 3 μL, Sac1 3 μL, plasmid 25 μL (concentration is 2 μg / μL), Buffer smart10 μL, ddH 2 Make up O to 100 μL; enzyme digestion process: time 8 h; temperature 37°C;

[0076] The steps of microinjection of mouse fertilized eggs were the same as in Example 1.

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Abstract

The invention provides a method for reducing ds DNA fragment tandems in CRISPR-Cas9 gene editing, and application thereof. The method comprises the following steps: connecting a suicide gene at a 5'end of a ds DNA, connecting a promoter of the suicide gene at a 3'end, mixing the ds DNA with a CRISPR-Cas9 reaction system, and transferring the mixture to a to-be-edited cell for completion of CRISPR-Cas9 gene editing. A suicide gene element and a promoter element thereof are connected to two ends of the ds DNA respectively, the promoter and the suicide gene are linked in series in case of a DNA tandem, and expression of the suicide gene is induced, so that the host cell is killed, death of the host cell is promoted, and the DNA tandem is removed.

Description

technical field [0001] The invention relates to the field of genetic engineering, and relates to a method for reducing the concatenation of double-stranded DNA fragments in CRISPR-Cas9 gene editing. Background technique [0002] Suicide gene refers to the introduction of certain viral or bacterial genes into target cells, and the expressed products are toxic substances or can catalyze the transformation of non-toxic precursors into toxic substances, thus causing the receptors carrying the gene to Cells are killed and such genes are called suicide genes. DTA and TK are two suicide genes commonly used in eukaryotic cells, among which DTA can produce toxic substances without adding substrates, and TK needs to add substrates to play a suicide role. At present, suicide genes are often used to treat tumors and infectious diseases, and are often used as negative screening in genetic engineering. [0003] With the in-depth study and interpretation of the two repair methods of non-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/89
CPCC12N15/89C12N15/907C12N2800/107
Inventor 官敏朱石磊林梓凡
Owner GEMPHARMATECH CO LTD
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