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Genome and epigenome engineering CRISPR toolkit for in vivo modulation of cis-regulatory interactions and gene expression in the chicken embryo.

Authors
  • Williams, Ruth M1
  • Senanayake, Upeka1
  • Artibani, Mara1, 2
  • Taylor, Gunes1
  • Wells, Daniel1
  • Ahmed, Ahmed Ashour1, 2, 3
  • Sauka-Spengler, Tatjana4
  • 1 University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK.
  • 2 University of Oxford, Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, UK.
  • 3 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women's Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK.
  • 4 University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK [email protected]
Type
Published Article
Journal
Development
Publisher
The Company of Biologists
Publication Date
Feb 23, 2018
Volume
145
Issue
4
Identifiers
DOI: 10.1242/dev.160333
PMID: 29386245
Source
Medline
Keywords
License
Unknown

Abstract

CRISPR/Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present an optimised, adaptable toolkit enabling genome and epigenome engineering in the chicken embryo, and demonstrate its utility by probing gene regulatory interactions mediated by neural crest enhancers. First, we optimise novel efficient guide-RNA mini expression vectors utilising chick U6 promoters, provide a strategy for rapid somatic gene knockout and establish a protocol for evaluation of mutational penetrance by targeted next-generation sequencing. We show that CRISPR/Cas9-mediated disruption of transcription factors causes a reduction in their cognate enhancer-driven reporter activity. Next, we assess endogenous enhancer function using both enhancer deletion and nuclease-deficient Cas9 (dCas9) effector fusions to modulate enhancer chromatin landscape, thus providing the first report of epigenome engineering in a developing embryo. Finally, we use the synergistic activation mediator (SAM) system to activate an endogenous target promoter. The novel genome and epigenome engineering toolkit developed here enables manipulation of endogenous gene expression and enhancer activity in chicken embryos, facilitating high-resolution analysis of gene regulatory interactions in vivo.

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