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.
Highlights
The functional annotation of the genome is a prerequisite to gain a deeper understanding of the molecular and cellular mechanisms that underpin development, homeostasis and disease of multicellular organisms
Since this previous proof-of principle study was restricted to testing pCFD6 with two single chimeric guide RNA (sgRNA) targeting the Wnt secretion factor Evenness interrupted (Evi, known as Wntless or Sprinter; Banziger et al, 2006; Bartscherer et al, 2006; Port and Bullock, 2016), we first tested whether this system is robust across target genes and tissues, a prerequisite to generate large-scale libraries of sgRNA strains targeting many or all Drosophila genes
The high performance of this resource relies on a) use of conditional sgRNA constructs to achieve strong dependency of Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) mutagenesis on Gal4, b) tunable Cas9 expression to achieve high on-target activity with low toxicity, c) the use of two sgRNAs targeting independent positions in the same gene to increase the fraction of cells that harbor non-functional mutations in both alleles
Summary
The functional annotation of the genome is a prerequisite to gain a deeper understanding of the molecular and cellular mechanisms that underpin development, homeostasis and disease of multicellular organisms. Forward genetic screens utilize random mutagenesis to introduce novel genetic variants, but are limited by the large number of individuals required to probe many or all genetic loci and difficulties in identifying causal variants. Reverse genetic approaches, such as RNA interference (RNAi), are gene-centric designed and allow to probe the function of a large number of genes (Boutros and Ahringer, 2008; Heigwer et al, 2018; Horn et al, 2011; Mohr et al, 2014). RNAi is often limited by incomplete penetrance due to residual gene expression and can suffer from off-target effects (Echeverri et al, 2006; Ma et al, 2006; Perkins et al, 2015)
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