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
Broad Institute of MIT and Harvard, Cambridge, MA, USA.
Center for System Biology, Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA.
Nat Genet. 2021 Aug;53(8):1166-1176. doi: 10.1038/s41588-021-00900-4. Epub 2021 Jul 29.
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.
对基因组功能和遗传变异进行有效解读，需要从顺式调控元件（CRE）的表观遗传图谱分析转向内源性功能的表征。我们开发了杂交链式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，可通过对天然转录本进行准确量化来表征经CRISPR干扰的CRE，同时还开发了CRISPR活性筛选分析（CASA），这是一种用于量化CRE活性的分层贝叶斯模型。通过超过32.5万次扰动，我们提供的证据表明，CRE可以调控多个基因，跳过最近的基因，并显示激活和/或沉默效应。在与胆固醇水平相关的FADS基因座上，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，从功能上确定因果变异，重要的是，识别它们的靶基因。
