{"doi":"10.1101/135525","title":"Genome and epigenome engineering CRISPR toolkit for probing\n                  <i>in vivo cis</i>\n                  -regulatory interactions in the chicken embryo","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  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\n                  <jats:italic>in vivo</jats:italic>\n                  .\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Summary Statement</jats:title>\n                  <jats:p>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.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>List of Abbreviations</jats:title>\n                  <jats:p>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).</jats:p>\n                </jats:sec>","journal":null,"year":null,"id":677833,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1771045,"name":"Upeka Senanayake","orcid":null,"position":1,"is_corresponding":false},{"id":669530,"name":"Mara Artibani","orcid":"0000-0002-4780-3199","position":2,"is_corresponding":false},{"id":1771050,"name":"Gunes Taylor","orcid":null,"position":3,"is_corresponding":false},{"id":1559011,"name":"Daniel Wells","orcid":"0000-0002-2007-8978","position":4,"is_corresponding":false},{"id":23320,"name":"Ahmed Ashour Ahmed","orcid":null,"position":5,"is_corresponding":false},{"id":50835,"name":"Tatjana Sauka-Spengler","orcid":"0000-0001-9289-0263","position":6,"is_corresponding":false},{"id":1771044,"name":"Ruth M Williams","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Genome and epigenome engineering CRISPR toolkit for probing\n                  <i>in vivo cis</i>\n                  -regulatory interactions in the chicken embryo","abstract":"<jats:title>Abstract</jats:title>\n                <jats:p>\n                  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\n                  <jats:italic>in vivo</jats:italic>\n                  .\n                </jats:p>\n                <jats:sec>\n                  <jats:title>Summary Statement</jats:title>\n                  <jats:p>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.</jats:p>\n                </jats:sec>\n                <jats:sec>\n                  <jats:title>List of Abbreviations</jats:title>\n                  <jats:p>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).</jats:p>\n                </jats:sec>","is_dataset_classified":null,"base_score":0.0,"endowment":0.0,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"21097893","pmcid":null,"openalex_id":"https://openalex.org/W2612185691","authors":[],"funders":[{"funder_name":"UK Research and Innovation","grant_id":"G0902418","title":"Weatherall Institute of Molecular Medicine - Application for an MRC-Oxford University Translational Medicine Institute"}],"total_grants":1,"fwci":null,"citation_percentile":null,"influential_citations":0,"citation_trend":[],"oa_status":"green","license":"https://www.biorxiv.org/about/FAQ#license","oa_locations":[{"url":"https://www.biorxiv.org/content/biorxiv/early/2017/05/11/135525.full.pdf","host_type":"repository"},{"url":"https://www.biorxiv.org/content/biorxiv/early/2017/05/11/135525.full.pdf","host_type":"repository"},{"url":"https://syndication.highwire.org/content/doi/10.1101/135525","host_type":"publisher"},{"url":"https://doi.org/10.1101/135525","host_type":"repository"},{"url":"https://dx.doi.org/10.1101/135525","host_type":""},{"url":"http://dx.doi.org/10.1101/135525","host_type":""}],"fields_of_study":["CRISPR and Genetic Engineering","RNA and protein synthesis mechanisms","RNA Interference and Gene Delivery","0301 basic medicine","03 medical and health sciences"],"mesh_terms":[],"keywords":["Epigenome","Biology","Enhancer","CRISPR","Cas9","Computational biology","Genome engineering","Genome editing","Genetics","Chromatin","Genome","Epigenetics","Regulation of gene expression","Gene","Transcription factor","Gene expression","DNA methylation"],"sdg_mappings":[],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-17T04:36:43.260237Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}