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A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide...

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Autores principales: Port, Fillip, Strein, Claudia, Stricker, Mona, Rauscher, Benedikt, Heigwer, Florian, Zhou, Jun, Beyersdörffer, Celine, Frei, Jana, Hess, Amy, Kern, Katharina, Lange, Laura, Langner, Nora, Malamud, Roberta, Pavlović, Bojana, Rädecke, Kristin, Schmitt, Lukas, Voos, Lukas, Valentini, Erica, Boutros, Michael
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062466/
https://www.ncbi.nlm.nih.gov/pubmed/32053108
http://dx.doi.org/10.7554/eLife.53865
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author Port, Fillip
Strein, Claudia
Stricker, Mona
Rauscher, Benedikt
Heigwer, Florian
Zhou, Jun
Beyersdörffer, Celine
Frei, Jana
Hess, Amy
Kern, Katharina
Lange, Laura
Langner, Nora
Malamud, Roberta
Pavlović, Bojana
Rädecke, Kristin
Schmitt, Lukas
Voos, Lukas
Valentini, Erica
Boutros, Michael
author_facet Port, Fillip
Strein, Claudia
Stricker, Mona
Rauscher, Benedikt
Heigwer, Florian
Zhou, Jun
Beyersdörffer, Celine
Frei, Jana
Hess, Amy
Kern, Katharina
Lange, Laura
Langner, Nora
Malamud, Roberta
Pavlović, Bojana
Rädecke, Kristin
Schmitt, Lukas
Voos, Lukas
Valentini, Erica
Boutros, Michael
author_sort Port, Fillip
collection PubMed
description Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.
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spelling pubmed-70624662020-03-11 A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila Port, Fillip Strein, Claudia Stricker, Mona Rauscher, Benedikt Heigwer, Florian Zhou, Jun Beyersdörffer, Celine Frei, Jana Hess, Amy Kern, Katharina Lange, Laura Langner, Nora Malamud, Roberta Pavlović, Bojana Rädecke, Kristin Schmitt, Lukas Voos, Lukas Valentini, Erica Boutros, Michael eLife Developmental Biology Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development. eLife Sciences Publications, Ltd 2020-02-13 /pmc/articles/PMC7062466/ /pubmed/32053108 http://dx.doi.org/10.7554/eLife.53865 Text en © 2020, Port 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
Port, Fillip
Strein, Claudia
Stricker, Mona
Rauscher, Benedikt
Heigwer, Florian
Zhou, Jun
Beyersdörffer, Celine
Frei, Jana
Hess, Amy
Kern, Katharina
Lange, Laura
Langner, Nora
Malamud, Roberta
Pavlović, Bojana
Rädecke, Kristin
Schmitt, Lukas
Voos, Lukas
Valentini, Erica
Boutros, Michael
A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title_full A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title_fullStr A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title_full_unstemmed A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title_short A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
title_sort large-scale resource for tissue-specific crispr mutagenesis in drosophila
topic Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062466/
https://www.ncbi.nlm.nih.gov/pubmed/32053108
http://dx.doi.org/10.7554/eLife.53865
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