Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology

A high-throughput drug screening technology, applied in the field of high-throughput sequencing, can solve problems such as skewed distribution of sgRNA libraries, low quality of sgRNA libraries, and a large number of PCR enzymes, so as to save screening costs and increase virus titers , to reduce the effect of false positives

Inactive Publication Date: 2017-02-15
TONGJI UNIV
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  • Summary
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many problems in international journal papers that the quality of sgRNA library is not high or the distribution of sgRNA library is skewed in the later stage of sgRNA sequencing.
In addition, the traditional library construction method is time-consuming and laborious, and requires a large amount of PCR enzymes, and the economic burden is relatively large.

Method used

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  • Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology
  • Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology
  • Method for screening drug target genes based on CRISPR/Cas9 high-throughput technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Screening of drug gene targets for breast cancer cell T47D. The specific method is as follows:

[0037] (1) sgRNA library design:

[0038] Use the websites http: / / crisprscan.org and http: / / www.e-crisp.org to design sgRNAs for gene groups of interest online, and design 10 sgRNAs for each gene.

[0039] (2) Establishment of sgRNA library by electroporation

[0040] The oligo fragment of the synthesized sgRNA was amplified by PCR and then ligated into the lentiCRISPRv2 plasmid by Gibson assembly. The ligation product was transformed into a competent state with a Bio-rad electroporator to obtain the sgRNA library.

[0041] (3) Packaging sgRNA library with lentivirus:

[0042] Under sterile conditions, culture 293FT cells, and use Roche's transfection reagent X-tremeGENE HP DNATransfection Reagent to package Lenti CRISPRv2 and two other virus packaging plasmids psPAX2 and pMD2.G into lentivirus. The medium was changed 15 hours after cell transfection, and the virus liqu...

Embodiment 2

[0053] CRISPR / Cas9 applied to the prostate cancer cell line LNcap is applied to a CRISPR / Cas9 screening method for large-scale screening of cancer gene targets, including the following steps:

[0054] (1) sgRNA library establishment:

[0055] Use the websites http: / / crisprscan.org and http: / / www.e-crisp.org to design and genome-wide sgRNA online. The sgRNA carrier uses Lenti CRISPRv2.

[0056] (2) Packaging sgRNA library with lentivirus:

[0057] Under sterile conditions, culture 293FT cells, and use Roche's transfection reagent X-tremeGENE HP DNATransfection Reagent to package Lenti CRISPRv2 and two other virus packaging plasmids psPAX2 and pMD2.G into lentivirus. Transfect 293FT cells according to the ratio of 4:3:1. Taking a 10cm culture dish as an example, the plasmid masses of Lenti CRISPRv2, psPAX2, and pMD2.G are 6μg, 4.5μg, and 1.5μg, respectively, and the amount of X-tremeGENE HP DNA Transfection Reagent 30 μL. The medium was changed 15 hours after cell transfecti...

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Abstract

The invention relates to a method for screening drug target genes based on a CRISPR/Cas9 high-throughput technology. The method comprises the steps of firstly establishing a sgRNA library; secondly packaging the sgRNA library by using slow viruses, and collecting the viruses; thirdly screening the sgRNA library in a cancer cell line; fourthly extracting cells obtained by screening and genome DNA of the cells before screening; and finally enriching sgRNA in genome DNA. Compared with the prior art, the method has the advantages that a CRISPR/Cas cell screening process is improved, the virus infection efficiency is determined with a simple and convenient method by utilizing puromycin resistance of infected cells, and MOI values of the viruses are determined; more importantly, a virus packaging method is greatly optimized, so that the virus packaging efficiency is improved to be more than 5 times that of a conventional method, and large-scale drug target screening cost can be greatly reduced; and the method is used for promoting industrialization of cancer drug target screening.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a method for screening drug target genes based on CRISPR / Cas9 high-throughput technology. Background technique [0002] Both oncogenes and tumor suppressor genes are potential targets of cancer gene therapy, so their identification has great application prospects in cancer treatment. Currently, drugs for the treatment of cancer have been developed targeting oncogenes EGFR, Alk and other genes. However, the current effective cancer gene-targeted drugs are far from meeting the needs of clinical treatment. Large-scale functional gene screening technology based on CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / clustered regularly interspaced short palindromic repeats associated protein 9), as an emerging technology in genetic engineering in recent years, can screen cancer-related genes at the genome-wide level, thereby providing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/113C40B50/06C12Q1/68
CPCC12N15/86C12N15/113C12N2740/15041C12Q1/6869C40B50/06C12Q2535/122C12Q2521/301
Inventor 蒋征刘小乐
Owner TONGJI UNIV
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