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Method for screening related host factors of target virus and application

A virus and target technology, applied in the field of large-scale screening of essential host factors or restrictive host factors of avian influenza virus, and preparation of mutant cell banks, can solve the problems of low repeatability, achieve high accuracy, strong controllability, and screening The method is simple and efficient

Active Publication Date: 2020-06-30
BEIJING DHELIXON BIOTECHNOLOGY CO LTD
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current large-scale screening of virus-associated host factors using CRISPR / Cas9 technology can only obtain a small number of candidate genes, and the reproducibility between different screens is low, which may be due to the CRISPR library, virus strains and analysis experiments. The choice of time point is different

Method used

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  • Method for screening related host factors of target virus and application
  • Method for screening related host factors of target virus and application
  • Method for screening related host factors of target virus and application

Examples

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Embodiment 1

[0052] Example 1 Technical process for large-scale preparation of mutant cell banks based on CRISPR technology

[0053] The technical process for the preparation of mutant cell bank in the present invention mainly includes the following four aspects (such as figure 1 shown):

[0054] First, use computer-assisted means to complete the sgRNA sequence design for the whole genome functional genes of model animals. Its main experimental method and key technology is to use computer-aided design combined with the latest bioinformatics technology to obtain an optimized list of sgRNA sequences targeting any DNA in the whole genome of animals.

[0055] Second, use chemical synthesis to establish a model animal-specific genome-wide sgRNA sequence library. Its main experimental methods and key technologies include using the obtained sgRNA sequence information to guide high-throughput DNA chemical synthesis and "assembling" plasmids after DNA synthesis into physical libraries that can be...

Embodiment 2

[0065] Example 2 Scale Screening of Avian Influenza Virus-Related Host Factors

[0066] The main steps of large-scale screening of avian influenza virus-related host factors in the present invention are as follows: Figure 4 As shown, the specific steps are as follows:

[0067] 1. A total of 112,227,891 sgRNAs were designed to extract 28,915 exons from 16,821 genes in the whole chicken genome. The lengths are all 20nt.

[0068] 2. Three kinds of plasmids were used: pCRISPR-sg6 plasmid library expressing gRNA and PB transposon; pCRISPR-S10 expressing Cas9 protein and PB transposon with neo drug screening tag; pCAG_PBase_4PB expressing PB transposase ,Such as Figure 5A , Figure 5B , Figure 5C shown.

[0069] 3. Prepare CRISPR / Cas9 library mutant cells, that is, the above three plasmids are co-transfected into DF1 cells, and the stably transfected cells are obtained after G418 selection for about 2 weeks; in addition, pCRISPR-S10 and pCAG_PBase_4PB are co-transfected int...

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Abstract

The invention discloses a method for screening related host factors of a target virus and an application, and belongs to the field of gene bank construction and application. A specific controllable mutant cell bank which covers whole genomes or partial functional genes is created by using a carrier system and a CRISPR library, the mutant cell bank is subjected to a challenge experiment of the target virus, surviving cells are collected, and an anti-virus mutant cell bank is obtained; and deep sequencing is performed on the cell bank to determine the related host factors of the target virus. The screening method is simple and efficient, can screen the related host factors of the target virus in the whole genome range, is high in accuracy, strong in controllability and low in cost, and can realize large-scale screening.

Description

technical field [0001] The invention relates to the technical field of CRISPR / Gas9, in particular to a method for large-scale screening of essential host factors or restrictive host factors of avian influenza virus by utilizing CRISPR libraries, preparation of mutant cell banks and other technical means. Background technique [0002] Bird flu is a disease caused by bird flu virus. After poultry is infected, it can manifest as respiratory disease and decreased egg production. In severe cases, it can lead to systemic disease and the mortality rate can reach 100%. The prevalence and outbreak of avian influenza will cause serious economic losses to the poultry industry. At the same time, avian influenza virus can also infect humans and other mammals, posing a huge threat to human life safety and public health. There are many subtypes of influenza virus, and antigenic drift and mutation will occur over time and space, and new subtypes will continue to evolve, so traditional avia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90C12Q1/6869
CPCC12N5/0611C12N15/902C07K14/465C12Q1/6869C12N2800/80C12N2810/10C12N2510/02
Inventor 吴森郭紫航
Owner BEIJING DHELIXON BIOTECHNOLOGY CO LTD
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