Gene editing method based on Vcre-Vloxp recombinase system and application of gene editing method

A technology of gene editing and recombinase, applied in the field of genetic engineering, can solve the problems of gene targeting failure, affecting technical accuracy, reducing the method of concatenation, etc., to achieve the effect of reducing the concatenation rate and improving the success rate of targeting

Active Publication Date: 2020-09-25
上海药康生物科技有限公司
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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

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  • Gene editing method based on Vcre-Vloxp recombinase system and application of gene editing method

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Embodiment 1

[0057] This example provides a gene editing method based on the Vcre-Vloxp recombinase system.

[0058] (1) Construction of double-stranded DNA (dsDNA):

[0059] The schematic diagram of the structure of the dsDNA constructed in this example is as follows figure 1 shown.

[0060] The dsDNA is shown as SEQ ID NO.5, wherein the promoter (Promoter) is a CMV promoter, and the terminator (Terminator) is a BGHpA terminator. Therefore, dsDNA can be recorded as CMV-Loxp-5×stop-Loxp-EGFP-Vloxp-BGHpA.

[0061] Construct the designed sequence on the plasmid, digest and purify to obtain dsDNA;

[0062] After digestion, the dsDNA was purified and recovered, and the dsDNA was diluted to 5 ng / μL with TE buffer.

[0063] SEQ ID NO.5 is shown below:

[0064]CGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTAT...

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Abstract

The invention provides a gene editing method based on the Vcre-Vloxp recombinase system and application of the gene editing method. The gene editing method includes the following steps that (1) a Vloxp sequence is connected to the 5' tail end or 3' tail end of a double-stranded DNA, or the Vloxp sequence is inserted between the functional regions of the double-stranded DNA; and (2) the double-stranded DNA, a Vcre protein and a gene editing reaction solution are mixed, and then transferred into a target cell to complete the gene editing. When the double-stranded DNA is repeatedly connected during an integration process, the double-stranded DNA is cut under the action of the Vcre protein, finally only one double-stranded DNA is left, no matter how many double-stranded DNAs are in tandem connection, the Vcre protein can cut all repetitive fragments from the first Vloxp sequence to the last Vloxp sequence, and therefore, the gene editing method can significantly reduce the tandem duplication phenomenon of recombinant DNA fragments in the integration process.

Description

technical field [0001] The invention relates to the field of genetic engineering, and relates to a gene editing method based on a Vcre-Vloxp recombinase system and an application thereof. Background technique [0002] The Cre-Loxp recombinase system is mainly composed of site-specific Cre recombinase and LoxP sequence, which was first discovered from P1 phage by Steinberg et al. Among them, Cre recombinase is a monomeric protein with 343 amino acids encoded by the P1 phage Cre gene, and belongs to the λInt enzyme supergene family. It not only has catalytic activity, but also is similar to restriction enzymes and can recognize specific DNA sequences, namely LoxP (locus of X(cross)-overinP1) sites. LoxP consists of two 13 bp inverted repeat sequences and an 8 bp spacer sequence in the middle. The 8 bp spacer sequence also determines the direction of LoxP. Cre recombinase can delete or recombine the gene sequence between LoxP sites,13 The inverted repeat of bp is the binding ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/89
CPCC12N15/85C12N15/89C12N2800/30C12N2999/007
Inventor 赵金龙朱石磊李雯婧林梓凡
Owner 上海药康生物科技有限公司
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