A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of the zygotic genome activation progrmme (bulk RNA-seq of arrayed CRISPRa validations and bulk RNA-seq of Patz1, Dppa2, Smarca5 and Carhsp1 cDNA overexpression)

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. Bulk RNA-seq of SAM mESCs after CRISPRa of Patz1, Dppa2, Dppa4, Smarca5, Pou2f2, Tsc22d4, Arnt, Sirt1, Smad1 and Carhsp1; bulk RNA-seq of E14 mESCs after 48h transfection with Dppa2, Smarca5, Patz1 and Carhsp1 GFP fusion constructs and FACS sorting of GFP+ cells. Each experiment was done in biological replicates.