生物学杂志

生物学杂志 ›› 2020, Vol. 37 ›› Issue (2): 1-.doi: 10.3969/j.issn.2095-1736.2020.02.001

• 研究报告 •    下一篇

在乳腺癌细胞中构建CRISPR多基因编辑系统

  

  1. 1. 江南大学 生物工程学院， 无锡 214122； 2. 江南大学 医学院， 无锡 214122
  • 出版日期:2020-04-18 发布日期:2020-04-17
  • 通讯作者: 戴晓峰，博士，教授，研究方向为乳腺癌细胞生物学，E-mail：xiaofeng.dai@jiangnan.edu
  • 作者简介:梅义，硕士研究生，研究方向为乳腺癌细胞生物学，E-mail：18801513880@163.com
  • 基金资助:
    江苏省自然科学基金项目（No: BK20161130）；江苏省六大人才高峰工程( No. SWYY-128 )；国家科技重大专项 (2018ZX10302205-004-002) 

Construction of a multiplexing gene modulation system in breast cancer cells

  1. 1. School of Biotechnology; 2. School of Medicine, Jiangnan University, Wuxi 214122, China
  • Online:2020-04-18 Published:2020-04-17

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摘要： CRISPR多基因编辑系统在动植物中已被广泛应用。为了在乳腺癌细胞中构建一套多基因编辑系统，利用内源性tRNA自剪切特点，将tRNA与小向导RNA(sgRNA)进行串联组合实现基因编辑，并在MCF7乳腺癌细胞中对KLF4、MYC、SOX2和OCT4 4个转录因子同时调控，在基因水平、蛋白表达以及功能改变3个层次验证该多基因编辑系统的功能性与可靠性。实验结果显示被调控的转录因子在细胞内的表达量提高2倍以上，侵袭能力明显增强，细胞的存活率提高50%，干细胞比例由2.57%提高到18.5%。同时，该系统在SUM149PT细胞系也被验证成功。综上，一套基于CRISPR的标准、精确、可复制、高效、可靠的多基因调控系统首次在乳腺癌细胞中构建成功。

关键词: 乳腺癌；CRISPR, 基因重编程

Abstract: With the CRISPR gene editing technology widely implemented in both plants and animals, a multi-gene editing system in breast cancer was constructed by harnessing the self-shearing characteristics of endogenous tRNA, integrating tRNA and multiple single guided RNA (sgRNA) targeting genes of interest into a single plasmid. Four transcription factors KLF4, MYC, SOX2 and OCT4 were modulated simultaneously using our system in breast cancer cell line MCF7 where the function and reliability of our system was verified from the transcriptional expression, protein expression and functional changes assays. The expressions of all modulated genes were upregulated above 2-fold, with the modulated cell displayed higher migration and invasive abilities and an increased cell availability by 50%. The proportion of stem cells was also increased from 2.57% to 18.5%. Concomitant multiplexing modulation of the same gene panel using our constructed system in breast cancer cell line SUM149PT was also achieved. Thus, based on CRSIPR, a standard, accurate, reproducible and efficient multiplexing gene modulation system in breast cancer cells was successfully built.

Key words: breast cancer, CRISPR, gene reprogramming

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