美国麻省理工学院和哈佛大学的布罗德研究所Ryan Tewhey研究组使用 HCR-FlowFISH直接表征顺式调控元件 (CRE)和复杂遗传关联的功能剖析。2021年7月29日出版的《自然—遗传学》发表了这项成果。

他们开发了杂交链反应荧光原位杂交结合流式细胞术 (HCR – FlowFISH)，这是一种广泛适用的方法，通过对天然转录本的准确量化来表征 CRISPR 扰动的CRE，以及 CRISPR 活性筛选分析 (CASA)，这是一种分层贝叶斯模型量化 CRE 活性。

在 > 325,000 次扰动中，他们用证据表明 CRE 可以调节多个基因，跳过最近的基因并显示激活和/或沉默效应。在胆固醇水平相关的 FADS 基因座上，他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，确定它们的靶基因。

据悉，对基因组功能和遗传变异的有效解释需要从CRE的表观遗传映射转变为内源性功能的表征。

附：英文原文

Title: Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR–FlowFISH

Author: Reilly, Steven K., Gosai, Sager J., Gutierrez, Alan, Mackay-Smith, Ava, Ulirsch, Jacob C., Kanai, Masahiro, Mouri, Kousuke, Berenzy, Daniel, Kales, Susan, Butler, Gina M., Gladden-Young, Adrianne, Bhuiyan, Redwan M., Stitzel, Michael L., Finucane, Hilary K., Sabeti, Pardis C., Tewhey, Ryan

Issue&Volume: 2021-07-29

Abstract: 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.

DOI: 10.1038/s41588-021-00900-4

Source: https://www.nature.com/articles/s41588-021-00900-4