利用CRISPRa和CRISPRi调控爱泼斯坦-巴尔病毒（EBV）阳性B细胞系中的基因表达。
Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi.
作者信息
Wang Liang Wei, Trudeau Stephen J, Wang Chong, Gerdt Catherine, Jiang Sizun, Zhao Bo, Gewurz Benjamin E
机构信息
Division of Medical Sciences, Harvard University, Boston, Massachusetts.
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts.
出版信息
Curr Protoc Mol Biol. 2018 Jan 16;121:31.13.1-31.13.18. doi: 10.1002/cpmb.50.
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.
爱泼斯坦-巴尔病毒（EBV）可将静止的原代小B细胞转化为能够在体外无限生长和存活的大型淋巴母细胞。这些细胞代表了肿瘤发生的一种模型。在本单元中，将在基因组DNA启动子和增强子元件的基因调控背景下讨论传统的成簇规律间隔短回文重复序列（CRISPR）的变体，即CRISPR激活（CRISPRa）和CRISPR干扰（CRISPRi）方法。用慢病毒转导稳定表达核酸酶缺陷型Cas9（dCas9）-VP64（与CRISPRa相关的Cas9）或dCas9-KRAB（与CRISPRi相关的Cas9）的淋巴母细胞B细胞系（LCL），该慢病毒编码靶向特定基因座的单向导RNA（sgRNA）。由dCas9分子及其同源sgRNA形成的核糖核蛋白复合物能够在感兴趣的启动子或增强子处进行序列特异性结合，从而影响由靶向启动子或增强子调控的基因的表达。©2018约翰威立父子公司。
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