CRISPR/Cas9 enrichment sequencing method applied in large-scale screening of cancer genes

A large-scale, cancer technology, applied in the field of high-throughput sequencing, which can solve the problems of time-consuming and laborious, a large number of PCR enzymes, and a large economic burden.

Inactive Publication Date: 2016-03-16
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, CRISPR/Cas9screen technology is only applied in a few laboratories at home and abroad, and has not been promoted
Moreover, this method requires high-throughput sequencing during use, because the template of sgRNA only accounts for a small part ...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Screening of cancer-related genes in lung cancer cell PC9. The specific method is as follows:

[0035] (1) sgRNA library establishment:

[0036] sgRNAs for cancer-related gene groups were designed online using the websites http: / / crisprscan.org and http: / / www.e-crisp.org. The sgRNA carrier uses LentiCRISPRv2.

[0037] (2) Packaging sgRNA library with lentivirus:

[0038] Under sterile conditions, culture 293FT cells, and use Roche's transfection reagent X-tremeGENEHPDNATransfectionReagent to package LentiCRISPRv2 and two other virus packaging plasmids psPAX2 and pMD2.G into lentivirus. Change the medium 15 hours after cell transfection, and collect the virus solution at the next 24 hours and 48 hours, mix the virus solution collected twice, and filter the virus solution with a 0.44μM filter to remove cell debris;

[0039] Taking a 10cm petri dish as an example, the plasmid masses of LentiCRISPRv2, psPAX2, and pMD2.G are 6 μg, 4.5 μg, and 1.5 μg, respectively, and th...

Embodiment 2

[0053] The CRISPR / Cas9 enrichment sequencing method applied to primary liver cancer cells for large-scale screening of cancer genes includes the following steps:

[0054] (1) sgRNA library establishment:

[0055] sgRNAs for cancer-related gene groups were designed online using the websites http: / / crisprscan.org and http: / / www.e-crisp.org. The sgRNA carrier uses LentiCRISPRv2.

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

[0057] Under sterile conditions, culture 293FT cells, and use Roche's transfection reagent X-tremeGENEHPDNATransfectionReagent to package LentiCRISPRv2 and two other virus packaging plasmids psPAX2 and pMD2.G into lentivirus. Transfect 293FT cells at a ratio of 4:3:1. Taking a 10 cm dish as an example, the plasmid masses of LentiCRISPRv2, psPAX2, and pMD2.G were 6 μg, 4.5 μg, and 1.5 μg, respectively, and the amount of X-tremeGENEHPDNATransfectionReagent was 30 μL. Change the medium 15 hours after cell transfection, and collect the virus solution ...

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PUM

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Abstract

The invention relates to a CRISPR/Cas9 enrichment sequencing method applied in large-scale screening of cancer genes. Firstly, an sgRNA library is established; then the sgRNA library is packaged by lentiviruses, and viruses are collected; the sgRNA library is screened in a cancer cell line, the obtained cells are extracted and screened, and genome DNAs of precancerous cells are screened; finally, enrichment of sgRNAs in the genome DNAs is carried out. When the provided method is compared with the prior art, the screening process of CRISPR/Cas9 cells is improved, the virus infection efficiency is determined and the virus MOI value is determined through a simple method and by utilization of puromycin resistance of infected cells, importantly, the restriction enzyme cutting method and a high flux method are combined, correct chromatin fragments can be obtained effectively, false positive caused by non-specific PCR amplification can be lowered, a DNA template of sgRNAs can be amplified efficiently, and a library establishment efficiency is raised.

Description

technical field [0001] The invention belongs to the technical field of high-throughput sequencing, and in particular relates to a CRISPR / Cas9 enrichment sequencing method applied to large-scale screening of cancer genes. 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. As an emerging technology in genetic engineering in recent years, large-scale functional gene screening technology based on CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / clustered regularly interspaced short palindromic repeats associated protein9) can screen cancer-related genes at the genome-wide level, thereby providing...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
Inventor 蒋征刘小乐李绪娟
Owner TONGJI UNIV
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