Unequivocal functional assessment of candidate genomic regulatory regions, such as transcriptional response elements, requires genetic alteration at their native chromosomal loci. Targeted DNA cleavage by Cas9 or other programmable nucleases enables analysis at virtually any genomic region, and diverse alleles generated by editing can be defined by deep sequencing for functional analysis. Interpretation of disrupted response elements, however, presents a special challenge, as these regions typically comprise clustered DNA binding motifs for multiple transcriptional regulatory factors (TFs); DNA sequence differences, natural or engineered, that affect binding by one TF can confer loss or gain of binding sites for other TFs. To address these and other analytical complexities, we created three computational tools that together integrate, in a single experiment, allele definition and TF binding motif evaluation for up to 9216 clones isolated, sequenced and propagated from Cas9-treated cell populations. We demonstrate 1) the capacity to functionally assess edited TF binding sites to query response element function, and 2) the efficacy and utility of these tools, by analyzing cell populations targeted by Cas9 for disruption of example glucocorticoid receptor (GR) binding motifs near FKBP5, a GR-regulated gene in the human adenocarcinoma cell line A549.

中文翻译：

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用于在基因组编辑的候选响应元素中定义等位基因和改变的调控基序的计算资源

候选基因组调控区域（例如转录反应元件）的明确功能评估需要在其天然染色体基因座进行遗传改变。Cas9 或其他可编程核酸酶的靶向 DNA 切割可以在几乎任何基因组区域进行分析，并且可以通过深度测序来定义编辑产生的各种等位基因以进行功能分析。然而，对被破坏的反应元件的解释提出了一个特殊的挑战，因为这些区域通常包含多个转录调节因子 (TF) 的聚集 DNA 结合基序；影响一种 TF 结合的天然或工程 DNA 序列差异可能会导致其他 TF 结合位点的丢失或获得。为了解决这些和其他分析复杂性，我们创建了三个计算工具，它们一起集成，在单个实验中，对从 Cas9 处理的细胞群中分离、测序和繁殖的多达 9216 个克隆进行等位基因定义和 TF 结合基序评估。我们展示了 1) 功能评估编辑的 TF 结合位点以查询响应元件功能的能力，以及 2) 这些工具的功效和实用性，通过分析 Cas9 靶向的细胞群以破坏 FKBP5 附近的示例糖皮质激素受体 (GR) 结合基序，人腺癌细胞系 A549 中的 GR 调节基因。