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

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 ...

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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.)

[0007] In the process of 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

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