Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated（Cas）protein 9（CRISPR/Cas9）的系統為基因敲除、敲入、活化、抑制和標記提供了精確的工具。在本次研究中，我們成功地使用CRISPR/Cas9敲除LM和HM20肺癌細胞中的癌症幹細胞相關基因ELAVL1（LM-ELAVL1-KO and HM20-ELAVL1-KO）。此外，為了進行有針對性的表觀遺傳修飾，我們使用dCas9 — 將Cas9核酸內切酶結構域突變，以產生核酸內切酶的功能喪失。dCas9與改變轉錄的蛋白質結構域融合，例如轉錄激活因子和組蛋白修飾因子。我們做出多個CRISPR-activated-ELAVL1的HM20克隆，像是HM20-dCas9VP64、HM20-dCas9VPR、HM20-dCas9p300 和HM20-dCas9SunTag。HuR（ELAVL1）是一種RNA的結合蛋白，和mRNA的穩定性和轉譯效率有關，也是調節細胞增生，生長和存活途徑的必要組成。我們發現ELAVL1敲除細胞（LM-ELAVL1-KO和HM20-ELAVL1-KO）的細胞遷移能力和增殖率顯著降低，而CRISPR / Cas9介導的表觀基因組編輯在ELAVL1激活細胞中這些功能有增加。有趣的是，細胞週期相關蛋白（如細胞週期蛋白B1）的表達在ELAVL1敲除細胞中減少，相反，這些基因在ELAVL1激活細胞中增加。我們可以通過CRISPR/Cas9介導的基因組和表觀基因組編輯來研究癌症幹細胞相關基因的功能。我們的結果可能為CRISPR/Cas9靶向癌症幹細胞相關基因的應用提供證據，作為一種新的治療策略，用於人類癌症治療。

Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated（Cas）protein 9 system provides a precision tool for gene knockin, knockout, activation, suppression and labeling. In this study, we successfully used CRISPR/Cas9 to knockout a cancer stem cell- related genes, ELAVL1, in LM and HM20 lung cancer cells（LM-ELAVL1-KO and HM20-ELAVL1- KO）. In addition, to make targeted epigenetic remodeling, researchers have mutated the nuclease domains of Cas9 to create a nuclease deficient（dCas9）. dCas9 is fused to a protein domain that alters transcription, such as a transcriptional activator and histone modifier. We generated several CRISPR-activated-ELAVL1 HM20 clones, including HM20-dCas9VP64, HM20-dCas9VPR, HM20-dCas9p300 and HM20-dCas9SunTag. HuR（ELAVL1）is a RNA-binding protein which modulates the stability and translational efficiency of mRNA encoding essential components of cellular proliferation, growth and survival pathways. We found that the cell migration ability and proliferation rate were significantly reduced in ELAVL1-knockout cells（LM-ELAVL1-KO and HM20-ELAVL1-KO）, whereas those functions were increased in ELAVL1-activation cells by CRISPR/Cas9-mediated epigenome editing. Interestingly, the expression of cell cycle-related proteins, such as cyclin B1, were reduced in ELAVL1-knockout cells, on the contrary, those genes were increased in ELAVL1-activation cells. We can investigate the functions of cancer stem cell- related genes by CRISPR/Cas9-mediated genome and epigenome editing. Our results may provide evidence for application of CRISPR/Cas9 targeting cancer stem cell-related genes, as a new treatment strategy, in human cancer therapy.

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