Human EpCAM gene knockout vector based on CRISPR-Cas9 system and cell strain thereof

By constructing a human EpCAM gene knockout vector based on the CRISPR-Cas9 system and transfecting colorectal cancer cell lines, the deficiencies in the study of the molecular mechanism of EpCAM in colorectal cancer were addressed, a cell line with low EpCAM expression was provided, and the research and treatment of colorectal cancer was promoted.

CN120758564APending Publication Date: 2025-10-10王丙萍 +1
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Patent Information

Application Number
CN202510982861.2
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2025-07-16
Publication Date
2025-10-10

AI Technical Summary

Technical Problem

The existing technology has not conducted in-depth research on the mechanism of action of EpCAM in colorectal cancer, and lacks effective gene knockout methods to study the molecular mechanism of EpCAM and colorectal cancer, which has affected the prevention, diagnosis and treatment of colorectal cancer.

Method used

A human EpCAM gene knockout vector based on the CRISPR-Cas9 system was constructed, including the pGMC00010 empty vector backbone structure and a specific sgRNA sequence. The vector was linked to the vector through enzyme cleavage sites to construct the EpCAM gene knockout vector, which was expressed in Escherichia coli. The colorectal cancer cell line HRT-18 was screened and transfected to identify transgenic cell lines with low EpCAM expression.

Benefits of technology

A colorectal cancer cell line with low expression of the EpCAM gene was provided, which was used to study the molecular mechanism of EpCAM and colorectal cancer, laying the foundation and providing new ideas and methods for the prevention, diagnosis and treatment of colorectal cancer.

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Abstract

The invention discloses three human EpCAM gene knockout vectors based on a CRISPR-Cas9 system and cell strains of the human EpCAM gene knockout vectors, and belongs to the technical field of gene engineering and biology. According to the invention, three pairs of sgRNAs (sgRNA-1 and sgRNA-2, sgRNA-3 and sgRNA-4 as well as sgRNA-5 and sgRNA-6) designed based on EpCAM genes are used, a U6 promoter is added in front of each sgRNA, each pair of sgRNAs is linked through T2A, each pair of sgRNAs is respectively connected to a pGMC00010 vector skeleton structure, and three knockout vectors pGMC-KO-EpCAM-1, pGMC-KO-EpCAM-2 and pGMC-KO-EpCAM-3 of the EpCAM genes are obtained. The three recombinant plasmids are respectively transformed into escherichia coli DH5alpha to obtain recombinant microorganisms. The gene knockout vector is used for respectively transfecting HRT-18 cells through a transfection reagent Lipo2000, a puromycin screening monoclonal method is used for obtaining positive cell strains, and finally flow cytometry is used for analyzing and screening to obtain transgenic cells HRT-18-KO-EpCAM-2 and HRT-18-KO-EpCAM-3 cell strains. According to the invention, the carrier with the EpCAM gene knocked out is constructed, and then the colorectal cancer cell strain with the corresponding low-expression EpCAM gene is constructed, which is very important for research on the molecular mechanism of generation and development of EpCAM and colorectal cancer.
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