Method for inserting exogenous fragments into DNA virus genome in efficient, fixed-point and directional manner

Abstract

The invention belongs to the technical field of virus gene recombination, and particularly relates to a method for inserting exogenous DNA fragments into a specific large DNA virus genome in a fixed-point, directional and efficient manner by utilizing a non-homologous recombination mechanism. The method comprises the following steps of 1) designing and constructing a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system for a specific cleavage site of the DNA virus genome; 2) designing and constructing an exogenous gene donor carrying one or more same cleavage sites; 3) introducing an expression vector of the CRISPR-Cas9 system and the exogenous gene donor into a specific cell according to a certain proportion and sequence; 4) aftera period of time, according to virus characteristics, selecting a proper infection time point and multiplicity of infection, and infecting the cell with viruses; 5) selecting an optimized time point to collect progeny recombinant viruses generated after virus infection; and 6) separating, screening and identifying target recombinant progeny DNA viruses. By means of the method, the recombination efficiency of inserting an exogenous gene into the DNA virus genome in a fixed-point and directional manner can be remarkably improved, so that the construction and screening time of the recombinant viruses can be shortened.

Description

technical field [0001] The invention belongs to the technical field of viral genome recombination, especially for double-stranded DNA viruses, and specifically relates to a high-fidelity method that does not rely on homologous recombination to efficiently and directionally insert foreign gene fragments into larger DNA virus genomes, which can be used It is widely used in the study of virus infection mechanism, exogenous gene expression, construction of viral vector, construction of attenuated vaccine and oncolytic virus, etc. Background technique [0002] There are two main methods for constructing recombinant DNA viruses: the first method is to stably preserve the entire DNA virus genome in Escherichia coli in the form of Bacterial Artificial Chromosome (BAC). In vitro, the gene modification operation is carried out by means of endonuclease, homologous recombination modification in Escherichia coli or gene integration system such as Cre-loxP. This recombination method can ...

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Problems solved by technology

However, due to the low frequency of homologous recombination, the efficiency of exogenous gene insertion by intracellular homologous recombination is very low, often lower than 0.1%, which will bring considerable problems to the construction of mutants or recombinant viruses. workload and rather long selection cycle

[0003] In the invention patent application with the application number CN201410041906.8 and the publication date of April 30, 2014 entitled "A Specific DNA Virus Genome Modification and Screening Method", a method that can efficiently convert exogenous The site-directed transformation method of gene insertion into the DNA virus genome, which uses the CRISPR-Cas9 system combined with the homologous recombination mechanism, significantly improves the efficiency of inserting foreign genes into the DNA virus genome (up to 8%), but the transformation method still has There is room for improvement, and for large fragments of foreign genes, the insertion efficiency often decreases with the increase of fragment length, so it cannot meet the needs of efficient insertion of foreign genes, especially large fragments of foreign genes

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