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CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity

The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vei...

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Autores principales: Xu, Xiang-Ru Shannon, Gantz, Valentino Matteo, Siomava, Natalia, Bier, Ethan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800851/
https://www.ncbi.nlm.nih.gov/pubmed/29274230
http://dx.doi.org/10.7554/eLife.30281
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author Xu, Xiang-Ru Shannon
Gantz, Valentino Matteo
Siomava, Natalia
Bier, Ethan
author_facet Xu, Xiang-Ru Shannon
Gantz, Valentino Matteo
Siomava, Natalia
Bier, Ethan
author_sort Xu, Xiang-Ru Shannon
collection PubMed
description The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ‘in locus’ mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.
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spelling pubmed-58008512018-02-08 CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity Xu, Xiang-Ru Shannon Gantz, Valentino Matteo Siomava, Natalia Bier, Ethan eLife Developmental Biology The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these ‘in locus’ mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis. eLife Sciences Publications, Ltd 2017-12-23 /pmc/articles/PMC5800851/ /pubmed/29274230 http://dx.doi.org/10.7554/eLife.30281 Text en © 2017, Xu et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Developmental Biology
Xu, Xiang-Ru Shannon
Gantz, Valentino Matteo
Siomava, Natalia
Bier, Ethan
CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title_full CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title_fullStr CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title_full_unstemmed CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title_short CRISPR/Cas9 and active genetics-based trans-species replacement of the endogenous Drosophila kni-L2 CRM reveals unexpected complexity
title_sort crispr/cas9 and active genetics-based trans-species replacement of the endogenous drosophila kni-l2 crm reveals unexpected complexity
topic Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800851/
https://www.ncbi.nlm.nih.gov/pubmed/29274230
http://dx.doi.org/10.7554/eLife.30281
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AT siomavanatalia crisprcas9andactivegeneticsbasedtransspeciesreplacementoftheendogenousdrosophilaknil2crmrevealsunexpectedcomplexity
AT bierethan crisprcas9andactivegeneticsbasedtransspeciesreplacementoftheendogenousdrosophilaknil2crmrevealsunexpectedcomplexity