The maternal-to-zygotic transition in mammals consist of the passing of the developmental control from the mother to the embryo and involves two major events: the depletion of maternal products and the activation of the zygotic genome. Zygotic genome activation, or ZGA, coincides with dramatic epigenetic reprogramming processes and is likely orchestrated by the interplay of epigenetic and transcriptional regulators. In this dissertation, I develop a novel high-throughput screening method that combines pooled CRISPR activation (CRISPRa) with single-cell transcriptomics and apply it to discover new epigenetic and transcriptional regulators of ZGA in mouse embryos.

In chapter 1, I introduce the biological context and latest technological advances related to CRISPR and single-cell transcriptomics. In chapter 2, I outline the materials and methods used in this dissertation. Chapters 3, 4 and 5 are a description and discussion of the results obtained. First, in chapter 3, I optimise the tools necessary to perform a CRISPRa screen with single-cell RNA-sequencing read-out. Specifically, I test CRISPRa in mouse embryonic stem cells (mESCs) using two well characterised markers of ZGA as targets and, subsequently, I develop and validate a method for detecting short guide RNA (sgRNA) molecules in single-cell RNA sequencing libraries.

In chapter 4, I select 230 screen candidates and construct a lentiviral sgRNA library to target their promoters by CRISPRa. After transducing this library into mESCs and performing single-cell RNA-sequencing for approximately 300,000 cells, I assess target gene activation by CRISPRa for each candidate gene and investigate different sgRNA features as well as several transcriptional and epigenetic parameters in relation to CRISPRa efficiency at the single-cell level.

In chapter 5, I discover 44 screen hits using unsupervised methods to identify latent sources of gene expression variation and differential gene expression analysis. Amongst the top hits, I identify the DNA binding protein Dppa2, the chromatin remodeller Smarca5 and the transcription factor Patz1. I subsequently validate these top hits using both CRISPRa and cDNA overexpression in mESCs. By doing this, I uncover new factors that promote a ZGA-like response in mESCs and are, therefore, strong candidates for ZGA regulation in vivo. Further molecular characterisation of Dppa2 and Smarca5 reveals that Smarca5 induces a ZGA-like transcriptional signature in mESCs via Dppa2, which in turns acts through the zygotic transcription factor Dux.

Lastly, in chapter 6, I summarise my findings, discuss their relevance in light of the literature in the field and propose future directions that arise from my conclusions.

Altogether, by developing and applying a new method for single-cell transcriptomic profiling of CRISPRa-perturbed cells, I provide novel system-level insights into the molecular mechanisms that orchestrate ZGA.