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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...
Autores principales: | , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2020
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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. |
format | Online Article Text |
id | pubmed-7062466 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
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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