Multiplexed engineering and analysis of endogenous enhancer activity in single cells: Bulk RNA-Seq validation

Transcriptional enhancers orchestrate cell-type specific gene expression programs critical to eukaryotic development, physiology, and disease. However, despite the large number of enhancers now identified, only a small number have been functionally assessed. Here, we develop MOsaic Single-cell Analysis by Indexed CRISPR Sequencing (Mosaic-seq), a method that measures one direct phenotype of enhancer repression: change of the transcriptome, at the single cell level. Using dCas9-KRAB to suppress enhancer function, we first implement a multiplexed system to allow the simultaneous measurement of the transcriptome and detection of sgRNAs by single cell RNA sequencing. We validate this approach by targeting the HS2 enhancer in the well-studied beta-globin locus. Next, through computational simulation, we demonstrate strategies to robustly detect changes in gene expression in these single cell measurements. Finally, we use Mosaic-seq to target 71 hypersensitive regions belonging to 15 super-enhancers in K562 cells by utilizing a lentiviral library containing 241 unique-barcoded sgRNAs. Our results demonstrate that Mosaic-seq is a reliable approach to study enhancer function in single cells in a high-throughput manner. Overall design: The overall design consists of RNA sequencing in bulk and in single K562 cells. As a control, we performed single-cell RNA sequencing of K562 cells expressing dCas9-KRAB and a control sgRNA targeting the non-expressed HSBP1 gene in two biological replicates. As a pilot project, we performed single cell RNA-Seq of dCas9-KRAB K562 cells after pooled infection of a library of 10 sgRNA viruses targeting promoters and enhancers in the beta-globin locus. As controls for this experiment, we individually infected these 10 sgRNAs and either performed 1) bulk RNA sequencing or 2) single cell RNA sequencing after pooling the separately infected cells. Finally, we scaled this single-cell approach to measure transcriptome changes for a library of 241 sgRNAs spanning 71 hypersensitive sites from 15 super-enhancers in K562 cells.

转录增强子（transcriptional enhancers）可精准调控细胞类型特异性的基因表达程序，这类程序对真核生物的发育、生理活动及疾病发生均发挥关键作用。尽管目前已鉴定出大量增强子，但仅有极少数得到了功能层面的验证。
本研究开发了基于索引CRISPR测序的镶嵌式单细胞分析技术（MOsaic Single-cell Analysis by Indexed CRISPR Sequencing, Mosaic-seq），该方法可在单细胞水平上检测增强子抑制的直接表型——转录组变化。研究借助dCas9-KRAB抑制增强子功能，首先构建了一套多重检测系统，可通过单细胞RNA测序（single-cell RNA sequencing）同时完成转录组分析与单向导RNA（single guide RNA, sgRNA）的检测。
本研究以研究较为透彻的β-珠蛋白基因座（beta-globin locus）中的HS2增强子为靶点，对该方法进行了验证。随后，通过计算机模拟，本研究确立了可在单细胞测序数据中稳定检测基因表达变化的分析策略。最后，本研究借助携带241条带有唯一条形码sgRNA的慢病毒文库（lentiviral library），针对K562细胞中15个超级增强子（super-enhancers）所包含的71个DNase I超敏位点（hypersensitive regions）开展Mosaic-seq检测。
研究结果证实，Mosaic-seq是一种可用于高通量单细胞水平增强子功能研究的可靠技术手段。
实验整体设计如下：整体方案涵盖K562细胞的批量RNA测序（bulk RNA sequencing）与单细胞RNA测序两类实验。作为对照组，本研究设置两组生物学重复，对表达dCas9-KRAB以及靶向非表达基因HSBP1的对照sgRNA的K562细胞开展单细胞RNA测序。
作为预实验，本研究对通过混合感染方式转染了靶向β-珠蛋白基因座启动子与增强子的10条sgRNA病毒文库的dCas9-KRAB阳性K562细胞开展单细胞RNA测序。为该预实验设置的对照包括：①分别感染上述10条sgRNA后开展批量RNA测序；②分别感染后将细胞混合，再开展单细胞RNA测序。
最后，本研究将该单细胞检测方法进行规模化拓展，针对K562细胞中来自15个超级增强子的71个超敏位点对应的241条sgRNA文库，检测其转录组变化。