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Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR–FlowFISH
Nature Genetics ( IF 31.7 ) Pub Date : 2021-07-29 , DOI: 10.1038/s41588-021-00900-4
Steven K Reilly 1, 2 , Sager J Gosai 1, 2, 3 , Alan Gutierrez 1 , Ava Mackay-Smith 1 , Jacob C Ulirsch 1, 3, 4 , Masahiro Kanai 1, 4, 5 , Kousuke Mouri 6 , Daniel Berenzy 6 , Susan Kales 6 , Gina M Butler 1 , Adrianne Gladden-Young 1 , Redwan M Bhuiyan 7, 8 , Michael L Stitzel 7, 8, 9 , Hilary K Finucane 1, 4 , Pardis C Sabeti 1, 2, 10, 11 , Ryan Tewhey 6, 12, 13
Affiliation  

Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR–FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.



中文翻译:


使用 HCR-FlowFISH 直接表征顺式调控元件和复杂遗传关联的功能解剖



对基因组功能和遗传变异的有效解释需要从顺式调控元件(CRE)的表观遗传图谱转向内源功能的表征。我们开发了杂交链式反应荧光原位杂交与流式细胞术 (HCR-FlowFISH) 的结合,这是一种广泛适用的方法,可通过准确定量天然转录本来表征 CRISPR 扰动的 CRE,同时还开发了 CRISPR 活性筛选分析 (CASA),这是一种分层贝叶斯模型,用于分析受 CRISPR 干扰的 CRE。量化 CRE 活动。在 >325,000 个扰动中,我们提供的证据表明 CRE 可以调节多个基因、跳过最近的基因并显示激活和/或沉默效果。在与胆固醇水平相关的FADS位点,我们将内源性筛选与报告基因检测相结合,以详尽地表征多个全基因组关联信号,在功能上指定因果变异,并且重要的是,识别其目标基因。

更新日期:2021-07-29
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