Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [4C]. Endogenous fine-mapping of functional regulatory elements in complex genetic loci in HAP1 [4C]

The majority of genetic loci linked to polygenic complex traits are found in non-coding regions of the human genome. These loci often exhibit complex gene regulatory relationships and linkage disequilibrium (LD) configurations, making it challenging to accurately identify causal variants and their target genes. We used multiplexed single-cell CRISPR interference and activation perturbations to investigate cis-regulatory element (CRE) and target gene expression relationships within tight LD in the endogenous chromatin context. We demonstrated the prevalence of multiple causality in perfect LD (pLD) for independent expression quantitative trait locus (eQTL) and uncovered fine-grained genetic effects on gene expression within pLD, which are difficult to decipher using traditional eQTL fine-mapping or existing computational methods. We found that over one third of the causal CREs lack classical epigenetic markers, and we functionally validated one of these hidden regulatory mechanisms. Leveraging Multiome single-cell epigenetic and sequence perturbations, we highlighted the regulatory plasticity of the human genome. Our study will guide the exploration of missing causal mechanisms in molecular trait formation and disease development. Overall design: Nine scRNA-seq datasets under varying multiplicity-of-infection (MOI) conditions, three single-cell multiome datasets (ATAC + Gene Expression) from modified HAP1 clones(dCas9-KRAB-blast, dCAS9-VP64-blast and Cas9-blast stably expressed HAP1 clones), four ATAC-seq datasets from wild-type and CRISPR-edited HAP1 cells(64bp deletion, 17bp deletion and CRISPRi around rs73156934-tagged cis-regulate element(CRE)), six CUT&TAG histone modification datasets and 1 Micro-C dataset from wild-type HAP1 cells, and three 4C-seq datasets for validating CRE-gene regulatory relationships in HAP1 cells.

绝大多数与多基因复杂性状相关的遗传位点定位于人类基因组的非编码区域。此类位点往往具备复杂的基因调控关系与连锁不平衡（linkage disequilibrium, LD）结构，使得精准鉴定因果变异及其靶基因的工作极具挑战。本研究采用多重单细胞CRISPR干扰与激活扰动技术，在内源染色质环境中探究紧密连锁不平衡区域内的顺式调控元件（cis-regulatory element, CRE）与靶基因的表达调控关系。我们证实，独立表达数量性状位点（expression quantitative trait locus, eQTL）的完全连锁不平衡（perfect LD, pLD）区域中普遍存在多因果性，并揭示了pLD区域内难以通过传统eQTL精细定位或现有计算方法解析的基因表达精细遗传效应。研究发现，超过三分之一的因果性顺式调控元件缺乏经典表观遗传标记，并对其中一种隐蔽调控机制完成了功能验证。借助单细胞多组学表观遗传与序列扰动技术，我们凸显了人类基因组的调控可塑性。本研究将为解析分子性状形成与疾病发生过程中缺失的因果机制提供重要指导。

整体实验设计：包含9组不同感染复数（multiplicity of infection, MOI）条件下的单细胞RNA测序（scRNA-seq）数据集；3组来自经改造的HAP1细胞株（分别稳定表达dCas9-KRAB-blast、dCAS9-VP64-blast及Cas9-blast）的单细胞多组学数据集（转座酶可及性测序ATAC-seq + 基因表达）；4组来自野生型及CRISPR编辑HAP1细胞的ATAC-seq数据集（涵盖64bp缺失、17bp缺失及rs73156934标记的顺式调控元件周围CRISPRi样本）；6组来自野生型HAP1细胞的CUT&TAG组蛋白修饰数据集与1组Micro-C染色质构象捕获数据集；另有3组用于验证HAP1细胞中CRE-基因调控关系的4C测序（4C-seq）数据集。