Massively parallel reporter assays and genome editing of ENCODE predicted regulatory elements

自2003年成立以来，DNA元素百科全书(ENCODE)联盟取得了令人瞩目的成就 人类基因组中所有功能元件的鉴定进展。然而，主要的限制是 目前目录的主要问题是，绝大多数元素尚未得到功能表征，其影响 关于它们功能的遗传变异的定义很模糊，它们激活或抑制的确切水平 他们之间的对话仍然没有被测量到，而且他们调控的特定基因(S)也不是明确的已知。致信地址 这些差距，我们将实施大规模并行功能分析，以表征超过100,000 基于编码的候选监管要素，以确认和量化其活动，并将许多 它们与它们的目标基因。关于上体与基因组大规模平行报道的系统比较 分析(MPRA)，我们表明附体分析无法准确地捕捉到调控活动的全部模式 在染色质的背景下观察到的。因此，我们只专注于测试候选对象的方法 在一个整合的、‘在基因组中’背景下的调控元素。首先，使用基于慢病毒的大规模并行报告 分析，我们将表征至少100,000个基于Encode的调控元件的启动子/增强子 整合到基因组中时的活性(lentiMPRA；目标1a)。重要的是，lentiMPRA可以在 几乎每种细胞类型，并利用慢病毒技术的持续发展。将使用早期结果 迭代地开发模型，以便为后续几轮功能表征做出更好的选择。 其次，我们将使用CRISPR/CAS9和多重同调定向修复来整合候选的子集 转录不活跃基因3‘非编码区的增强子，使我们能够进一步验证和表征它们的 在自然基因组环境中激活转录的能力(‘在基因组中’Starr-seq；目标1b)。最后，我们会 实施新的范例，包括基于CRISPR/Cas9的多重基因组编辑，然后是RNAseq/ ATAC-SEQ分子图谱用于表征全基因组范围的候选调控元件子集 他们的天然基因组背景下突变对他们的功能后果，同时也决定了 他们调控的靶基因(S)(大规模并行基因组编辑；目标2)。虽然我们最初会集中我们的 除了在K562和HepG2细胞上的努力外，我们还将根据需要在其他细胞系上进行工作 编码联盟，我们25%的容量专门用于一组公共元素。结合了 在其他功能鉴定中心的努力下，我们的工作将提供史无前例的基因组验证 以及ENCODE定义的候选监管元素的特征，同时还有助于深入了解我们的 了解基因调控的基本生物学以及调控变异如何导致人类疾病 风险。

Since its inception in 2003, the Encyclopedia of DNA Elements (ENCODE) Consortium has made remarkable progress towards the identification of all functional elements in the human genome. However, major limitations of the current catalog are that the vast majority of elements have not been functionally characterized, the impact of genetic variation on their function is poorly defined, the precise levels of activation or repression that they confer remain unmeasured, and the specific gene(s) that they regulate are not definitively known. To address these gaps, we will implement ‘in genome’ massively parallel functional assays to characterize over 100,000 ENCODE-based candidate regulatory elements, to confirm and quantify their activities as well as to link many of them to their target genes. In a systematic comparison of episomal vs. genomic massively parallel reporter assays (MPRA), we show that episomal assays fail to accurately capture the full patterns of regulatory activity that are observed in the context of chromatin. We therefore focus exclusively on methods that test candidate regulatory elements in an integrated, ‘in genome’ context. First, using lentivirus-based massively parallel reporter assays, we will characterize at least 100,000 ENCODE-based regulatory elements for their promoter/enhancer activity while integrated into the genome (lentiMPRA; Aim 1a). Importantly, lentiMPRA can be carried out in almost every cell type and leverages ongoing developments in lentivirus technology. Early results will be used to iteratively develop models that make better selections for subsequent rounds of functional characterization. Second, we will use CRISPR/Cas9 and multiplex homology directed repair to integrate a subset of candidate enhancers to the 3’ UTR of transcriptionally inactive genes, allowing us to further validate and characterize their ability to activate transcription in a natural genomic context (‘in genome’ STARR-seq; Aim 1b). Finally, we will implement a new paradigm involving CRISPR/Cas9-based multiplex genome editing followed by RNAseq/ ATAC-seq molecular profiling to characterize a genome-wide subset of candidate regulatory elements in their native genomic context for the functional consequences of mutations on them, while also determining the target gene(s) that they regulate (massively parallel genome editing; Aim 2). Although we will initially focus our efforts on K562 and HepG2 cells, we will also perform work in other cell lines as appropriate for the needs of the ENCODE Consortium, with 25% of our capacity dedicated to a common set of elements. Combined with the efforts of the other functional characterization centers, our work will provide unprecedented ‘in genome’ validation and characterization of ENCODE-defined candidate regulatory elements, while also facilitating insights into our understanding of the basic biology of gene regulation and how regulatory variants contribute to human disease risk.