A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
- German Cancer Research Center (DKFZ) and Heidelberg University, Germany
Abstract
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.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
Deutsche Forschungsgemeinschaft (TRR186)
- Fillip Port
- Bojana Pavlovic
- Michael Boutros
European Research Council (Decode)
- Michael Boutros
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Port et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 15,302
- views
-
- 1,887
- downloads
-
- 130
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
A large-scale resource for tissue-specific CRISPR mutagenesis in Drosophila
eLife 9:e53865.
https://doi.org/10.7554/eLife.53865
Further reading
-
- Developmental Biology
During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory toward the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.
-
- Developmental Biology
- Neuroscience
The ligand Netrin mediates axon guidance through a combination of haptotaxis over short distances and chemotaxis over longer distances.
