CRISPR activation screening at single-cell transcriptomic resolution: discovering new regulators of zygotic genome activation
type: CreativeWork
description: 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…
abstract: 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…
author: Alda Catalinas, Celia
language: en
keywords: CRISPR, CRISPRa, single-cell, scRNA-seq, epigenetics, development, pre-implantation development, zygotic genome activation, ZGA, maternal proteins, maternal deposits, oocyte, 2C embryo, two-cell embryo, 2C-like cells, mouse embryonic stem cells, mESCs, ESCs, transcription, chromatin, Dppa2, Smarca5, Patz1, MERVL, Zscan4, CROP-seq, screen, 10X Genomics, ZGA-like, Dux
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/c3a9b561-a4b2-44b2-9e8b-c936006a5c20/download
license: https://creativecommons.org/licenses/by-nc-sa/4.0/
date published: 2020-05-16
date modified: 2026-07-22T22:24:04Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.48119