Zygotic genome activation (ZGA) is an essential transcriptional event in embryonic development that coincides with extensive epigenetic reprogramming. Complex manipulation techniques and maternal stores of proteins preclude large-scale functional screens for ZGA regulators within early embryos. Here, we combined pooled CRISPR activation (CRISPRa) with single-cell transcriptomics to identify regulators of ZGA-like transcription in mouse embryonic stem cells, which serve as a tractable, in vitro proxy of early mouse embryos. Using multi-omics factor analysis (MOFA+) applied to ∼200,000 single-cell transcriptomes comprising 230 CRISPRa perturbations, we characterized molecular signatures of ZGA and uncovered 24 factors that promote a ZGA-like response. Follow-up assays validated top screen hits, including the DNA-binding protein Dppa2, the chromatin remodeler Smarca5, and the transcription factor Patz1, 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 both system-level and molecular insights into the mechanisms that orchestrate ZGA.

合子基因组激活 (ZGA) 是胚胎发育中的一个重要转录事件，与广泛的表观遗传重编程相吻合。复杂的操作技术和母体蛋白质储存排除了对早期胚胎中 ZGA 调节器的大规模功能筛选。在这里，我们将合并的 CRISPR 激活 (CRISPRa) 与单细胞转录组学相结合，以鉴定小鼠胚胎干细胞中 ZGA 样转录的调节因子，作为一种易于处理的体外早期小鼠胚胎的代表。使用多组学因子分析 (MOFA+) 应用于约 200,000 个单细胞转录组，包括 230 个 CRISPRa 扰动，我们表征了 ZGA 的分子特征，并发现了促进 ZGA 样反应的 24 个因子。后续分析验证了顶级筛选命中，包括 DNA 结合蛋白Dppa2、染色质重塑剂 Smarca5和转录因子Patz1，功能实验表明Smarca5对 ZGA 样转录的调节依赖于Dppa2。总之，我们对 CRISPRa 扰动细胞的单细胞转录组分析为协调 ZGA 的机制提供了系统水平和分子方面的见解。