A single-cell transcriptomics CRISPR-activation screen identifies new epigenetic regulators of zygotic genome activation

Abstract

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 processes 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 to systematically probe candidate regulators of ZGA. We screened 230 epigenetic and transcriptional regulators by upregulating their expression with CRISPRa in mouse embryonic stem cells (ESCs). Through single-cell RNAsequencing (scRNA-seq) of CRISPRa-perturbed cells, we generated approximately 200,000 single-cell transcriptomes, each transduced with a unique short-guide RNA (sgRNA) targeting a specific candidate gene promoter. Using integrative dimensionality reduction of the perturbation scRNA-seq profiles, we characterized molecular signatures of ZGA and uncovered 44 factors that promote a ZGA-like response in ESCs, both in the coding and non-coding transcriptome. Upon upregulation of these factors, including the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1, ESCs adopt an early embryonic-like state. Supporting their roles as ZGA regulators, Dppa2 and Smarca5 knock-out ESCs lose expression of ZGA genes, however, overexpression of Dppa2 in Smarca5 knock-out ESCs, but not vice versa, rescues ZGA-like expression, suggesting that Smarca5 regulates ZGA upstream and via Dppa2. Together, our single-cell transcriptomic profiling of CRISPRaperturbed cells provides comprehensive system-level insights into the molecular mechanisms that orchestrate ZGA. Highlights• First large-scale screen combining pooled CRISPRa with scRNA-seq. • Multi-omics factor analysis identifies a ZGA-like signature for 44 of the candidate regulators. • Dppa2, Smarca5 and Patz1 were validated as strong inducers of ZGA gene expression. • Smarca5 regulates zygotic genome activation in a Dppa2-dependent manner.

Maternal SMARCA5 is required for major ZGA in mouse embryos

“…Overall, the above results reveal that SMARCA5 is a key regulator of the activation of the genome in two-cell stage embryos in vivo, as was hypothesised following the results from the CRISPR activation (CRISPRa) screen in mESCs in vitro (Alda-Catalinas et al, 2019).…”

Massively Parallel CRISPR‐Based Genetic Perturbation Screening at Single‐Cell Resolution

“…[ 6c ] A ZGA‐like transcriptional signature is elicited by TFs and epigenetic regulators, as discovered by activation‐based scCRISPR in mouse ESCs. [ 54 ] Additionally, scCRISPR elucidated the mRNA splicing pattern of the receptors for activated C kinase 1 and protein tyrosine phosphatase receptor type C in combination with nanopore sequencing. [ 28 ] Additionally, using scATAC‐seq, scCRISPR screenings revealed epigenetic landscape remodelers in human B lymphocytes and leukemia cells, [ 29,30,55 ] and they verified non‐coding drivers of estrogen receptor‐dependent carcinogenic regulatory networks.…”

“…However, the scCRISPR experiment is still unaffordable for most laboratories because of the high cost of single‐cell devices and reagents. [ 11 b] Limited to the number of single cells prepared with devices, each perturbation covers a small number of cells so that one perturbation can't be fully “exposed,” [ 54 ] which results in the variation of sgRNA efficiency and low signal‐to‐noise ratio in readout data. Besides, preparing of single‐cell sample is quite difficult and requires harsh conditions for sample processing and preservation.…”

Single-cell functional genomics of natural killer cell evasion in blood cancers

“…To generate lentiviral sgRNA libraries for single-cell CRISPR screens, guides targeting screen hits (three sgRNAs for each gene) and non-targeting control sgRNAs (four for K562, six for other screens; Table S6A) were cloned into CROPseq-Guide-Puro (a gift from Christoph Bock, Addgene # 86708) (Datlinger et al, 2017) or into CROP-sgRNA-MS2 (a gift from Wolf Reik, Addgene # 153457) (Alda-Catalinas et al, 2020). Lentivirus was produced and cells were transduced as described above using different concentrations of virus.…”