CRISPR-Mediated Multiplexed Epigenomic Perturbation of Putatively Functional ASoC Sites in Human Neural Progenitor Cell Model at Single-cell Resolution

Functional interpretation of noncoding disease variants, which likely regulate gene expression, has been challenging. Chromatin accessibility (openness) strongly influences gene expression in neurodevelopment; however, to what extent genetic variants can alter chromatin openness in the context of brain disorders/traits is largely unknown. Using human induced pluripotent stem cell (iPSC)-derived neurons as a neurodevelopmental model, we identified abundant open-chromatin regions absent in brains and thousands of genetic variants exhibiting allele-specific open-chromatin (ASoC). ASoC variants are overrepresented in brain enhancers, transcription-factor-binding sites, and quantitative-trait-loci affecting histone modifications or DNA methylation. ASoC variants are also highly enriched for those associated with brain disorders/traits. Following-up schizophrenia-associated ASoC variants by multiplex epigenomic perturbation, computational fine-mapping, and CRISPR-editing further confirmed their regulatory activities and cis-targeted genes. Our study provides the first snapshot of neuronal ASoC landscape and a framework for prioritizing functional disease variants. Overall design: NPC samples were derived from individual cell line #05, #07, and #08. Collected NPC samples were processed with 10x Genomics Chromium Single Cell 3' Reagent Kits v2 according to the manufacturer's instructions. Sequencing of the prepared library was performed on an Illumina sequencer generating approximately 450M reads in total.

对于大概率参与基因表达调控的非编码疾病变异，其功能注释长期以来都是极具挑战性的研究课题。染色质可及性（即染色质开放程度）对神经发育过程中的基因表达具有显著调控作用；然而，在脑疾病或脑相关性状的背景下，遗传变异能够在多大程度上改变染色质开放程度，目前仍尚未明确。本研究以人类诱导多能干细胞（human induced pluripotent stem cell, iPSC）分化得到的神经元作为神经发育模型，鉴定出了大量在脑组织中不存在的开放染色质区域，以及数千个表现出等位基因特异性开放染色质（allele-specific open-chromatin, ASoC）的遗传变异。ASoC变异在脑增强子、转录因子结合位点以及影响组蛋白修饰或DNA甲基化的数量性状基因座（quantitative trait loci, QTL）中显著富集，同时也显著富集于与脑疾病或脑相关性状相关的遗传变异中。通过多重表观基因组扰动、计算精细定位以及CRISPR编辑技术对精神分裂症相关的ASoC变异进行后续验证，进一步确认了其调控活性及其顺式靶基因。本研究首次绘制了神经元中ASoC的全景图谱，并为功能性疾病变异的优先级排序提供了研究框架。
整体实验设计：神经前体细胞（neural progenitor cells, NPC）样本来自#05、#07和#08三个独立细胞系。收集的NPC样本按照制造商说明书使用10x Genomics Chromium单细胞3'端试剂试剂盒v2进行处理。构建好的文库在Illumina测序仪上进行测序，总测序量约为4.5亿条读段。