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Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo

Ruth M Williams, Upeka Senanayake, Mara Artibani, Gunes Taylor, Daniel Wells, Ahmed Ashour Ahmed, Tatjana Sauka-Spengler
doi: https://doi.org/10.1101/135525
Ruth M Williams
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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Upeka Senanayake
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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Mara Artibani
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
2University of Oxford, Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, UK
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Gunes Taylor
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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Daniel Wells
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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Ahmed Ashour Ahmed
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
2University of Oxford, Ovarian Cancer Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, OX3 9DS, UK
3Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Women’s Centre, John Radcliffe Hospital, Oxford, OX3 9DU, UK
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Tatjana Sauka-Spengler
1University of Oxford, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Oxford, OX3 9DS, UK
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  • For correspondence: tatjana.sauka-spengler@imm.ox.ac.uk
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Abstract

CRISPR-Cas9 genome engineering has revolutionised all aspects of biological research, with epigenome engineering transforming gene regulation studies. Here, we present a highly efficient 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 efficient guide-RNA expression from novel chick U6-mini-vectors, provide a strategy for rapid somatic gene knockout and establish 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.

Summary Statement: We present an optimised toolkit for efficient genome and epigenome engineering using CRISPR in chicken embryos, with a particular focus on probing gene regulatory interactions during neural crest development.

List of Abbreviations: Genome Engineering (GE), Epigenome Engineering (EGE), single guide RNA (sgRNA), Neural Crest (NC), Transcription Factor (TF), Next Generation Sequencing (NGS), somite stage (ss), Hamburger Hamilton (HH).

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted May 08, 2017.
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Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo
Ruth M Williams, Upeka Senanayake, Mara Artibani, Gunes Taylor, Daniel Wells, Ahmed Ashour Ahmed, Tatjana Sauka-Spengler
bioRxiv 135525; doi: https://doi.org/10.1101/135525
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Genome and epigenome engineering CRISPR toolkit for probing in vivo cis-regulatory interactions in the chicken embryo
Ruth M Williams, Upeka Senanayake, Mara Artibani, Gunes Taylor, Daniel Wells, Ahmed Ashour Ahmed, Tatjana Sauka-Spengler
bioRxiv 135525; doi: https://doi.org/10.1101/135525

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