A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of the zygotic genome activation programm (10X Genomics CRISPRa screen dataset)

Zygotic genome activation (ZGA) is a crucial developmental milestone that remains poorly understood. This first essential transcriptional event in embryonic development coincides with extensive epigenetic reprogramming and is orchestrated, in part, by the interplay of transcriptional and epigenetic regulators. Here, we developed a novel high-throughput screening method that combines pooled CRISPR-activation (CRISPRa) with single-cell transcriptomics and applied this method to systematically probe candidate regulators of ZGA-like transcription. We screened 230 epigenetic and transcriptional factors by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNA-sequencing (scRNA-seq), we generated approximately 200,000 single-cell transcriptomes of CRISPRa-perturbed cells, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using multi-omics factor analysis (MOFA) of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. We further validated nine candidate genes by arrayed CRISPRa analysed by bulk transcriptomics, which demonstrates that the combination of CRISPRa with scRNA-seq is a powerful and valid approach to identify regulators of ZGA-like transcription. Additional cDNA overexpression assays for three top hits, the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, confirmed these factors as ZGA-like regulators by alternative methods. Supporting these findings, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes and functional experiments revealed that Smarca5’s regulation of ZGA-like transcription is dependent on Dppa2. Together, our single-cell transcriptomic profiling of CRISPRa-perturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA.

合子基因组激活（Zygotic Genome Activation, ZGA）是一个至关重要的发育里程碑，但其背后的调控机制至今仍未被充分阐明。这一胚胎发育过程中的首个核心转录事件，与大规模表观遗传重编程同步发生，且部分由转录调控因子与表观遗传调控因子的相互作用所介导。本研究开发了一种将池化CRISPR激活（CRISPR-activation, CRISPRa）技术与单细胞转录组学相结合的新型高通量筛选方法，并将其用于系统探究类合子基因组激活转录的候选调控因子。我们在小鼠胚胎干细胞（embryonic stem cells, ESCs）中通过CRISPRa上调230种表观遗传与转录因子的表达，完成了筛选。借助单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq），我们获得了约20万个经CRISPRa扰动的细胞的单细胞转录组数据，每个细胞均被靶向特定候选基因启动子的唯一短向导RNA（short-guide RNA, sgRNA）转导。通过对扰动后的scRNA-seq图谱进行多组学因子分析（multi-omics factor analysis, MOFA），我们解析了ZGA的分子特征，并鉴定出24种可在小鼠ESCs中调控编码与非编码转录组、诱导类ZGA应答的因子。我们进一步通过阵列式CRISPRa结合批量转录组学分析，对9个候选基因进行了验证，结果证实CRISPRa与scRNA-seq联用是鉴定类ZGA转录调控因子的高效可靠方法。此外，我们还通过替代实验方法，对3个筛选位列前茅的候选基因——DNA结合蛋白Dppa2、染色质重塑因子Smarca5以及转录因子Patz1进行了cDNA过表达验证，进一步确认了这些因子作为类ZGA调控因子的身份。相关基因敲除实验结果亦支持上述结论：Dppa2与Smarca5敲除的ESCs会丧失ZGA相关基因的表达；功能实验进一步揭示，Smarca5对类ZGA转录的调控依赖于Dppa2。综上，本研究通过对CRISPRa扰动细胞的单细胞转录组分析，为解析合子基因组激活的调控分子机制提供了全面的系统层面认知。