Large-Scale In Vivo Functional Characterization of the Human Cistrome

 描述(申请人提供)：数以千计的顺式元件的体外功能已经用大规模平行报告分析进行了表征。然而，顺式元件功能的体内表征一直局限于使用小鼠模型的少数哺乳动物增强剂。最近，全基因组CRISPR/Cas9基因敲除筛选已被证明是评估基因组DNA功能的一种有效方法，并为在体内大规模研究顺式元件功能提供了机会。我们已经开发了一个统计模型来预测gRNA效率，用于CRISPR/Cas9筛选的优化，以及一个用于分析筛选数据的计算管道。此外，我们在几个细胞系中进行了全基因组CRISPR/Cas9基因敲除筛选试验，并确定了对细胞生长至关重要的已知基因。我们通过对蛋白质-DNA相互作用和染色质可及性的全基因组分析，展示了在研究基因转录调控方面的专业知识。在这项提议中，我们假设同时的慢病毒传递 在一个顺式调控元件两侧的两个引导RNA(GRNA)中，可以有效地敲除(KO)该元件，CRISPR/Cas9 KO筛选是大规模人类顺式元件体内功能表征的有效方法。我们建议发展高通量Cstrome CRISPR/Cas9缺失筛查的实验和计算方法，以阐明哺乳动物体内顺式元件功能的调控机制，并扩大我们对正常生理和疾病中转录调控的认识。具体地说，我们建议1)使用CRISPR/Cas9基因敲除屏幕来确定八个人类细胞系中对细胞生长有强烈影响的转录因子和染色质调节子；2)对可能的顺式调控元件进行CRISPR/Cas9基因敲除筛选，以确定对基因表达和细胞生长或存活有强烈影响的元件；3)计算模拟活体细胞周期蛋白的功能，实验验证模型并创建Cistrome注释网络服务器。在这些研究的结论中，我们将发展实验技术和计算工具，继续研究体内顺式元件的功能，并扩大我们对细胞特异性基因转录调控机制的了解。由此产生的资源将改进对疾病相关体细胞突变或非编码区生殖系变异的功能的解释。

 DESCRIPTION (provided by applicant): The in vitro functions of thousands of cis-elements have been characterized using massively parallel reporter assays. However, the in vivo characterization of cis-element functions has been limited to a handful of mammalian enhancers using mouse models. Recently, genome-wide CRISPR/Cas9 knockout screening has proven to be an efficient approach for assessing the function of genomic DNAs and presenting an opportunity to study in vivo cis-element functions on a large scale. We have developed a statistical model to predict gRNA efficiency for the optimization of CRISPR/Cas9 screens, as well as a computational pipeline for the analysis of the screen data. In addition we have performed pilot genome- wide CRISPR/Cas9 knockout screens in several cell lines and have identified known genes essential cell growth. We have demonstrated expertise in studying gene transcriptional regulation through genome-wide analysis of protein-DNA interactions and chromatin accessibility. In this proposal, we hypothesize that the simultaneous lentiviral delivery of two guide RNAs (gRNA) flanking a cis-regulatory element can efficiently knockout (KO) the element, and that CRISPR/Cas9 KO screens are an efficient approach for the large-scale in vivo functional characterization of human cis-elements. We propose to develop the experimental and computational approaches for high-throughput cistrome CRISPR/Cas9 deletion screens to elucidate the regulatory mechanisms of mammalian cis-element in vivo functions and expand our knowledge on transcriptional regulation in normal physiology and diseases. Specifically we propose to 1) use CRISPR/Cas9 knockout screens to identify transcription factors and chromatin regulators in eight human cell lines that have strong effect on cell growth; 2) conduct CRISPR/Cas9 knockout screens on putative cis-regulatory elements to identify elements with strong effects on gene expression and cell growth or survival; 3) computationally model in vivo cistrome function, experimentally validate the model and create a Cistrome annotation web server. At the conclusion of these studies, we will have developed the experimental techniques and computational tools for continued investigation of in vivo cis-element functions, and expanded our knowledge on the mechanism of cell-specific gene transcriptional regulation. The resulting resource will improve interpretation of the function of disease associated somatic mutations or germline variants in the non-coding regions.