Although neural tube defects (NTDs) are common in humans, little is known about their multifactorial genetic causes. While most mouse models involve NTDs caused by a single mutated gene, we have previously described a multigenic system involving susceptibility to NTDs. In mice with a mutation in Cecr2, the cranial NTD exencephaly shows strain-specific differences in penetrance, with 74% penetrance in BALB/cCrl and 0% penetrance in FVB/N. Whole genome linkage analysis showed that a region of chromosome 19 was partially responsible for this difference in penetrance. We now reveal by genetic analysis of three subinterval congenic lines that the chromosome 19 region contains more than one modifier gene. Analysis of embryos showed that although a Cecr2 mutation causes wider neural tubes in both strains, FVB/N embryos overcome this abnormality and close. A microarray analysis comparing neurulating female embryos from both strains identified differentially expressed genes within the chromosome 19 region, including Arhgap19, which is expressed at a lower level in BALB/cCrl due to a stop codon specific to that substrain. Modifier genes in this region are of particular interest because a large portion of this region is syntenic to human chromosome 10q25, the site of a human susceptibility locus.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. eLife digest Twenty years after the release of the sequence of the human genome, the role of many genes is still unknown. This is partly because some of these genes may only be active in specific types of cells or for short periods of time, which makes them difficult to study. A powerful way to gather information about human genes is to examine their equivalents in ‘model’ animals such as fruit flies. Researchers can use genetic methods to create strains of insects where genes are deactivated; evaluating the impact of these manipulations on the animals helps to understand the roles of the defunct genes. However, the current methods struggle to easily delete target genes, especially only in certain cells, or at precise times. Here, Port et al. genetically engineered flies that carry CRISPR-Cas9, a biological system that can be programmed to ‘cut’ and mutate precise genetic sequences. The insects were also manipulated in such a way that the CRISPR elements could be switched on at will, and their quantity finely tuned. This work resulted in a collection of more than 1,700 fruit fly strains in which specific genes could be deactivated on demand in precise cells. Further experiments confirmed that this CRISPR system could mutate target genes in different parts of the fly, including in the eyes, gut and wings. Port et al. have made their collection of genetically engineered fruit flies publically available, so that other researchers can use the strains in their experiments. The CRISPR technology they refined and developed may also lay the foundation for similar collections in other model organisms. Introduction 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. Drosophila melanogaster has provided many fundamental insights into metazoan biology, in particular in the form of systematic gene discovery through genetic screens. 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. In contrast, 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). In addition, RNAi reagents can be genetically encoded and used to screen for gene function with spatial and temporal precision (Dietzl et al., 2007; Kaya-Çopur and Schnorrer, 2016; Ni et al., 2009). However, 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). While genetic screens have contributed enormously to our understanding of gene function, large parts of eukaryotic genomes remain not or only poorly characterized (Brown et al., 2009; Dickinson et al., 2016; White et al., 2013). For example, in Drosophila only 20% of genes have associated mutant alleles (Kaufman, 2017). Therefore, there exists an urgent need to develop innovative approaches to gain a more complete understanding of the functions encoded by the various elements of the genome. Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) - CRISPR-associated (Cas) systems are adaptive prokaryotic immune systems that have been adopted for genome engineering applications (Doudna and Charpentier, 2014; Wang et al., 2016). Cas9 complexed with a single chimeric guide RNA (sgRNA) mediates site-specific DNA double strand breaks and subsequent DNA repair can result in small insertions and deletions (indels) at the break point. However, not all Cas9-mediated indel mutations abrogate gene function. To compensate for that, strategies have been developed to introduce simultaneously several mutations in the same gene. The efficiency of such multiplexing strategies has been demonstrated in flies, mice, fish and plants, and several sgRNAs are often required to generate bi-allelic loss-of function mutations in all cells (Port and Bullock, 2016; Xie et al., 2015; Yin et al., 2015). Furthermore, to gain a comprehensive understanding of the often multifaceted functions genetic elements have in multicellular organisms requires methods that enable spatial or temporal control of gene disruption. To restrict CRISPR mutagenesis to defined cells, tissues or developmental stages, specific regulatory regions are commonly employed to drive Cas9 expression. However, Cas9 expression vectors with tissue-specific enhancers often display ‘leaky’ Cas9 expression in other tissues and poor control of CRISPR mutagenesis has been observed in multiple systems, including flies, mice and patient derived xenografts (Chen et al., 2017; Dow et al., 2015; Hulton et al., 2019; Port and Bullock, 2016). It has recently been demonstrated that expressing both Cas9 and sgRNA from conditional regulatory elements can result in tightly controlled genome editing (Port and Bullock, 2016), but the robustness of such a strategy across many genomic target sites has so far not been explored. Here, we describe a large-scale resource for spatially restricted mutagenesis in Drosophila. The system mediates robust mutagenesis across target genes, giving rise to a large fraction of cells containing gene knock-outs and displays tight spatial and temporal control. We developed a series of tunable Cas9 lines that allow gene editing with high efficiency and low toxicity independent of enhancer strength. These can be used with a growing library of sgRNA transgenes, which currently comprise over 1700 Drosophila strains, for systematic mutagenesis in any somatic tissue or the germline. Furthermore, we present the first large-scale transgenic CRISPR screen using this resource, which confirms its high efficiency and specificity and reveals multiple uncharacterized genes with essential, but unknown function. Results Robust tissue-specific CRISPR mutagenesis We set out to develop a large-scale resource that would allow systematic CRISPR-mediated gene disruption with tight spatial and temporal control (Figure 1A). In Drosophila, tissue-specific expression of transgenes is most commonly performed via the binary Gal4/UAS system (Brand and Perrimon, 1993) and thousands of Gal4 lines with specific temporal and spatial expression patterns are publicly available. To harness this resource for tissue-specific CRISPR mutagenesis we aimed to utilize UAS-Cas9 transgenes and combine them with the sgRNA expression vector pCFD6, which enables Gal4-dependent expression of sgRNA arrays. We have previously shown that conditional expression of both Cas9 and sgRNAs is necessary to achieve tight control of mutagenesis (Figure 1B; Port and Bullock, 2016). Since this previous proof-of principle study was restricted to testing pCFD6 with two sgRNAs targeting the Wnt secretion factor Evenness interrupted (Evi, also known as Wntless or Sprinter; Bänziger 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. To this end, we created various transgenic fly lines harbouring a pCFD6 transgene encoding two sgRNAs targeting a single gene at two independent positions. These were crossed to flies containing a UAS-cas9.P2 transgene and a tissue-specific Gal4 driver. We then analysed if mutations were efficiently induced, restricted to the appropriate cells and caused the expected phenotypes. We observed efficient and specific gene disruption in wing imaginal discs with pCFD6 sgRNA transgenes targeting the Drosophila beta-Catenin homolog armadillo (arm, Figure 1C), as well as the transcription factor senseless (sens) or the transmembrane protein smoothened (smo) (Figure 1—figure supplement 1A,B). To test tissue-specific CRISPR mutagenesis in a different tissue context, we targeted Notch (N) in the Drosophila midgut, which is derived from the endoderm. We observed a strong increase in stem cell proliferation and an accumulation of cells with small nuclei, which matches the described phenotype of N mutant clones in the midgut (Ohlstein and Spradling, 2006; Figure 1D and Figure 1—figure supplement 2). Interestingly, we observed a qualitative difference between perturbation of N expression by RNAi, which only induces hyperplasia in female flies (Figure 1—figure supplement 2; Hudry et al., 2016; Siudeja et al., 2015), and N mutagenesis by CRISPR, which induces strong overgrowth in both male and female midguts (Figure 1—figure supplement 2). We also tested conditional mutagenesis of neuralized (neur) and yellow (y) along the dorsal midline and of sepia (se) in the developing eye and observed in each case the described null mutant phenotype in the expected domain (Figure 1E,F, Figure 1—figure supplement 1C). Figure 1 with 2 supplements see all Download asset Open asset Conditional CRISPR mutagenesis with pCFD6 is robust across target genes and tissues. (A) Schematic overview of the workflow. To perform tissue-specific targeted mutagenesis flies transgenic for a specific Gal4 driver (X-Gal4) and UAS-Cas9 are crossed to flies with a UAS-sgRNA transgene. Offspring from this cross express Cas9 and sgRNAs in Gal4 expressing cells, leading to mutagenesis of the target gene. (B) Schematic of gene editing outcomes typically observed with a single, ubiquitous sgRNA (lower left) or a conditional array of several sgRNAs (lower right). Leaky expression, that is expression in the absence of Gal4, from conditional Cas9 transgenes gives rise to ectopic mutagenesis in combination with ubiquitous, but not conditional, sgRNAs. Gene editing in tissues typically results in genetic mosaics, which can be enriched for bi-allelic knock-out cells through sgRNA multiplexing. (C) Conditional CRISPR mutagenesis in wing imaginal discs with nub-Gal4 in the wing pouch. Gene editing with pCFD6-arm2x results in loss of Arm protein exclusively in the Gal4 expression domain in nearly all cells. Control animals express the nub-Gal4 driver and UAS-cas9.P2. Scale bar = 50 µm. (D) Conditional CRISPR mutagenesis of Notch in intestinal stem cells drives tumor formation in the midgut. esgts (esg-Gal4 tub-Gal80ts) was used to repress expression of UAS-cas9.P2 and pCFD6-N2x until adult stages. Mutagenesis was induced for 5 days at 29°C and flies were returned to 18°C to avoid Cas9.P2 mediated toxicity. Posterior midguts 15 days after induction of mutagenesis are shown. esgts UAS-cas9.P2 pCFD6-N2x tissue shows an accumulation of stem cells (DNA marked in cyan) and an increase in mitotic cells (pHistone3 in magenta). Quantification of phenotypes are shown in Figure 1—figure supplement 2. Control genotype is esgts UAS-cas9.P2 pCFD6-se2x. Scale bar = 50 µm. (E) Mutagenesis of neur in pnr-Gal4 UAS-cas9.P2 pCFD6-neur2x animals results in loss of thoracic bristles along the dorsal midline, where pnr-Gal4 is expressed. Note the tissue patch that retains bristles, reflecting mosaic mutagenesis. (F) Mutagenesis of the pigmentation gene se in the eye. GMR-Gal4 UAS-casp.P2 pCFD6-se2x animals develop a uniform dark eye coloration. Control animals in (E) and (F) express the respective Gal4 driver and UAS-cas9.P2 pCFD6-Sfp24C12x. (G) pCFD6 mediated mutagenesis in the germline. Shown is a summary of the mutational status at each sgRNA target site in individual F1 flies. nos-Gal4VP16 UAS-cas9.P1 pCFD6 flies expressing sgRNAs targeting the indicated essential genes are viable, demonstrating germline restricted mutagenesis, and transmit mutant alleles to their offspring. Shown is a summary of the mutational status at each sgRNA target site in individual flies. All lines, except the one targeting Dpp (asterisk), transmit mutant alleles to the majority of offspring. Flies expressing sgRNAs targeting Dpp in the germline produce few viable offspring and transmitted only a single, in-frame, mutation out of 11 analysed offspring. The same sgRNA construct results in highly efficient mutagenesis in somatic tissues (see Figure 4), consistent with haploinsufficiency of Dpp in the Drosophila embryo. Next, we tested whether pCFD6-sgRNA2x also mediates efficient mutagenesis in the germline, where some UAS vectors are silenced (DeLuca and Spradling, 2018; Huang et al., 2018). This is a particularly important application, as it allows to create stable and sequence-verified mutant fly lines, which can be backcrossed to remove potential off-target mutations. We crossed previously described nos-Gal4VP16 UAS-Cas9.P1 flies (Port et al., 2014) to sgRNA strains targeting either neur, N, cut (ct), decapentaplegic (dpp) or Ras85D. Despite the fact that all five genes are essential for Drosophila development and act in multiple tissues, nos-Gal4VP16 UAS-Cas9.P1 pCFD6-sgRNA2x flies were viable and morphologically normal, demonstrating tightly restricted mutagenesis. We then tested their offspring for CRISPR induced mutations at the sgRNA target sites. Crosses with pCFD6-sgRNA2x targeting neur, N, ct and Ras85D passed on mutations to most or all analysed offspring (Figure 1G). Mutations were often found on both target sites, were frequently out-of-frame and included large deletions of 8 and 14 kb between the sgRNA target sites (Figure 1G). In contrast, nos-Gal4VP16 UAS-Cas9.P1 pCFD6-dpp2x flies produced only few viable offspring of which only 1/11 carried a mutation, which was in-frame. Since dpp is known to be haploinsufficient (St Johnston et al., 1990), this is consistent with a high number of dpp loss-of function alleles being transmitted to the next generation. Together, these experiments demonstrate that sgRNA expression from pCFD6 mediates efficient and tightly restricted mutagenesis in various somatic cell types as well as the germline and establishes that tissue-specific CRISPR mutagenesis in Drosophila is robust across genes and tissues. Tunable Cas9 expression to balance activity and toxicity We and others have shown that expression of high amounts of Cas9 protein is toxic in various organisms (Jiang et al., 2014; Poe et al., 2019; Port et al., 2014; Yang et al., 2018). For example, overexpression of Cas9 in the wing imaginal disc of nub-Gal4 UAS-cas9.P2 animals results in a strong induction of apoptosis (Figure 2—figure supplement 1A). Since only relatively low levels of Cas9 are sufficient for efficient gene editing (Figure 2—figure supplement 1B), we sought to engineer a system that would allow to tune Cas9 expression to optimally balance activity and toxicity. Such a system would ideally allow to modulate Cas9 levels independent of enhancer strength, in order to be compatible with the wide range of available Gal4 lines. We employed a method that uses upstream open reading frames (uORF) of different length to predictably reduce translation of the main, downstream ORF (Ferreira et al., 2013; Kozak, 2001; Southall et al., 2013). We created a series of six UAS-cas9 plasmids containing uORFs of different length, ranging from 33 bp (referred to as UAS-uXSCas9) to 714 bp (UAS-uXXLCas9, Figure 2A). When combined with nos-cas9 these plasmids resulted in Cas9 protein levels inversely correlated with the length of the uORF (Figure 2B, Figure 2—figure supplement 1C). Reducing the amount of Cas9 protein resulted in a strong decrease in the number of apoptotic cells (Figure 2C). Importantly, three UAS-uCas9 transgenes with moderate levels of Cas9 expression and apoptosis levels similar to controls did mediate full on-target gene editing activity at the evi locus in wing imaginal discs (Figure 2D, Figure 2—figure supplement 1C). Together, these experiments demonstrate that the UAS-uCas9 vector series enables titration of Cas9 expression to avoid toxicity without sacrificing gene editing activity. Figure 2 with 2 supplements see all Download asset Open asset A transgenic series for tunable Cas9 expression to balance activity and toxicity. (A) Principle of the UAS-uCas9 series. Translation of the downstream ORF is inversely correlated with length of the upstream ORF in bicistronic mRNAs. The UAS-uCas9 series consists of transgenes that harbor uORFs of different length to modulate expression of Cas9. (B - D) Systematic characterization of Cas9 expression, toxicity and mutagenesis efficiency of the UAS-uCas9 series. Transgenes of the UAS-uCas9 series were recombined with nub-Gal4 and crossed to the apoptosis sensor UAS-GC3Ai (B, C) or pCFD6-evi2x (D). Graphs show data as individual dots, and boxplots as a data summary, with the line representing the median and the box the interquartile range. (B) Quantification of anti-Cas9 staining intensity in wing discs of the indicated genotype. Cas9 levels gradually reduce as the size of the uORF increases. N ≥ 6 wing discs. (C) Elevated levels of apoptosis were only observed with UAS-uXSCas9. The longest uORF (uXXL) encodes EGFP, preventing visualization of dying cells with GC3Ai. Quantification of fluorescent intensity of the GC3Ai reporter in the wing pouch. N ≥ 14 wing discs. (D) All transgenes of the UAS-uCas9 series mediate evi mutagenesis, with transgenes containing the four shortest uORFs (XS-L) leading to comparable gene editing that removes Evi from nearly all cells in the Gal4 expression domain. Quantification of staining intensity for Evi protein in the wing pouch (Gal4 on), relative to Evi staining in the hinge region (Gal4 off). N ≥ 6 wing discs. (E, E’) CRISPR mutagenesis patterns reflect Gal4 expression history. (E) Fluorescence of GFP, which turns over, reflects most recent Gal4 expression pattern. (E’) CRISPR mutagenesis, visualized by activation of the CIGAR reporter, is permanent and reveals the Gal4 expression history. Images of a representative wing disc are shown to the left of each panel and average intensity projection of several discs registered to a common template are shown on the right (see Materials and methods). Areas that are CIGAR positive in many discs appear bright, while areas devoid of signal in most discs appear dark. (F, F’) Incomplete repression of CRISPR mutagenesis by temperature-sensitive Gal80. (F) Principle of the Gal80ts system. At 18°C Gal80 binds and inhibits Gal4. (F’) Mutagenesis is still observed at 18°C in 11/24 discs and observed preferentially in the Gal4 expression domain, indicating incomplete Gal4 suppression by Gal80ts. (G - G’’) Control of CRISPR mutagenesis by a flip-out cassette. (G) In the absence of FLP recombinase a FRT-flanked GFP flip-out cassette (FRT sites represented by triangles) separates Cas9 from the promoter, resulting in cells that express GFP, but no Cas9. In the presence of FLP, the GFP cassette is excised and Cas9 is expressed. (G’) Staining for the transcription factor Cut reveals a continuous stripe of cells expressing ct along the dorsal-ventral boundary in wildtype tissue. (G’’) A pulse of FLP expression was used to excise the GFP flip-out cassette in a subset of cells (marked by the absence of GFP). Cut expression (magenta) is exclusively lost in GFP negative cells. Scale bar = 50 µm. Next, we generated a toolbox of various fly strains harbouring a UAS-uMCas9 transgene and a Gal4 driver on the same chromosome (Figure 2—figure supplement 2A,B). Such stocks can be crossed to transgenic sgRNA lines to induce conditional CRISPR mutagenesis in Gal4-expressing cells. We tested the spatial mutagenesis pattern for a number of novel Gal4 UAS-uMCas9 lines in the wing imaginal disc of third instar larva by either visualizing the loss of protein encoded by the target gene with a specific antibody, or by using the transgenic CIGAR reporter (Brunner et al., 2019). CIGAR encodes an ubiquitously expressed fluorescent protein that is only efficiently translated once an upstream sequence has been mutated by CRISPR gene editing (Brunner et al., 2019). While not all CRISPR-mediated mutations lead to induction of the fluorophore encoded by CIGAR, this strategy has the advantage that it readily reveals CRISPR activity throughout the entire organism. We found that while some Gal4 UAS-uMCas9 lines resulted in mutagenesis exclusively in cells positive for Cas9 at that stage (Figure 2—figure supplement 2D,E), others had much broader mutagenesis patterns (Figure 2E, Figure 2—figure supplement 2F,G). For example, in third instar wing discs ptc-Gal4 is expressed in a narrow band of cells along the anterior-posterior boundary (Figure 2E). However, CRISPR mutagenesis with ptc-Gal4 frequently leads to mutations throughout the entire anterior compartment (Figure 2E’), likely reflecting broader expression of ptc-Gal4 in early development or expression at low level in this domain. Similar effects were observed with dpp-Gal4 (Figure 2—figure supplement 2G). Therefore, additional regulatory mechanisms to temporally control Cas9 expression are highly desirable when using Gal4 lines with dynamic expression patterns during development. We first employed the temperature-sensitive Gal80 repressor to suppress Gal4 activity. While Gal80ts mediated strong inhibition of mutagenesis in ptc-Gal4 UAS-uMCas9 tub-Gal80ts flies at the restrictive temperature of 18°C, we still observed mutagenesis in Gal4-expressing cells in 11/24 wing discs, indicating residual Gal4 activity (Figure 2F). We therefore tested an alternative strategy to induce CRISPR mutagenesis at a given time point. We created a transgene that harbors a FRT-flanked GFP Stop-cassette between the UAS promoter and the uMCas9 expression cassette (UAS-FRT-GFP-FRT-uMCas9, Figure 2G). A brief pulse of Flp recombinase (from a hs-Flp transgene) can be used to excise the GFP cassette at the desired time and induce Cas9 expression. We validated this approach by mutagenizing ct in a negatively marked subset of cells in the wing disc and observed loss of Ct protein exclusively in cells that had lost GFP expression (Figure 2G). These experiments highlight the need to critically evaluate spatial mutagenesis patterns in conditional CRISPR experiments and suggest strategies for additional control of gene editing in cases where the Gal4 expression pattern alone does not suffice. We envision that in the future other systems for conditional transgene expression, such as the chemical-dependent GeneSwitch system (Osterwalder et al., 2001; Roman et al., 2001), split-Gal4 (Luan et al., 2006) or conditional transgene degradation (Sethi and Wang, 2017) will also be combined with CRISPR to further refine mutagenesis patterns. A large-scale transgenic sgRNA library Having established the robustness of our method and developed an optimised Cas9 toolkit, we next focused our efforts on the generation of a large-scale sgRNA resource. First, we generated and validated three sgRNA lines targeting genes with highly restricted expression patterns, which can be used as controls for effects of Cas9/sgRNA expression and induction of DNA damage in the majority of tissues where their target gene is not expressed (Figure 3—figure supplement 1; Graveley et al., 2011). To allow systematic screening of functional gene groups we then designed sgRNAs against all Drosophila genes encoding transcription factors, kinases and phosphatases, as well as a large number of other genes encoding fly orthologs of genes implicated in human pathologies (Figure 3A, see Materials and methods). We used CRISPR library designer (Heigwer et al., 2016) to compile a list of all sgRNAs that do not have predicted off-target sites elsewhere in the genome. We then selected sgRNAs depending on the position of their target site within the target gene. We chose sgRNAs targeting coding exons shared by all mRNA isoforms and target sites that were located in the 5’ half of the open reading frame, where indel mutations often have the largest functional impact. We then grouped sgRNAs in pairs, with each pair targeting sites typically separated by approximately 500 bp of protein coding DNA (see Materials and methods). Next, we devised an efficient cloning protocol to insert defined sgRNA pairs into pCFD6. This utilized synthesized oligonucleotide pools, which allow cloning of hundreds to thousands of sgRNA plasmids in parallel in a single tube, followed by clonal selection of individual pCFD6-sgRNA2x plasmids and sequence validation (Figure 3B, see Materials and methods). We also generated a derivative of pCFD6, pCFD6.FRT, which harbors incompatible FRT2 and FRT5 sites before and after the sgRNA cassette, respectively. These recombination sites can be used to exchange sequences either side of the sgRNA cassette, for example the promoter, or to add additional sgRNAs to the array (Figure 3C). We validated that both FRT sites mediate highly efficient chromosome exchange in vivo (Figure 3D). We then generated a large-scale transgenic sgRNA library, which we collectively refer to as the ‘Heidelberg CRISPR Fly Design Library’ (short HD_CFD library). This growing resource currently contains 2622 plasmids and 1739 fly stocks targeting 1513 unique genes (Supplementary file 1). Fly lines are so far available for 545/754 (72%) transcription factors, 199/230 (87%) protein kinases and 141/207 (68%) phosphatases (Figure 2D). Figure 3 with 1 supplement see all Download asset Open asset Generation of a large-scale sgRNA library. (A) Design of the sgRNA pairs used for the HD_CFD library. sgRNAs were designed through CLD and filtered to target common exons in the 5’ORF and not overlap the start codon. sgRNAs were then paired to target two independent positions in the same gene. As an example the locations of the two target sites in ovo targeted by the two sgRNAs encoded in line HD_CFD000172 is shown. Exons are represented as boxes and regions in blue are protein coding. (B) Experimental strategy for the generation of the transgenic sgRNA library. sgRNA target sequences are encoded on oligonucleotides synthesized and cloned in pool. Individual plasmids are sequence verified and transformed into Drosophila at attP40 on the second chromosome following a pooled injection protocol followed by genotyping of individual transformants. (C) Applications of the pCFD6::FRT vector. pCFD6::FRT contains two non-compatible FRT sites either side of the sgRNA cassette. Using compatible FRT sides in trans allows to exchange sequences upstream or downstream of the sgRNAs in vivo. (D) Efficient promoter or sgRNA exchange in vivo. Summary of FLP/FRT mediated exchange of the sgRNA promoter (left) or sgRNAs (right). Each line represents a single sequenced animal. Red and blue boxes either side of the triangle (representing FRT) indicate successful recombination. (E) Summary statistics of the different functional groups present in the sgRNA library. Given is the number of genes from each category that are covered by fly lines, plasmids or against which currently no tools are available. Note that for some genes two fly lines or plasmids exist. Status in September 2019 is shown. Group ‘Others’ contains mainly genes with human orthologs associated with cancer development in humans. HD_CFD sgRNA lines mediate efficient mutagenesis and allow robust CRISPR screening To test on-target activity of HD_CFD sgRNA strains, we crossed a random selection of 28 HD_CFD lines to an act-cas9;;tub-Gal4/TM3 strain, which is expected to mediate ubiquitous mutagenesis in combination with active sgRNAs. We then sequenced PCR amplicons encompassing the sgRNA target sites (see Materials and methods) and analysed editing efficiency by ICE analysis (Hsiau et al., 2019). We found that the vast majority (26/28) of HD_CFD sgRNA lines resulted in gene editing on both target sites (Figure 4A). For 12/28 of lines editing on both sites was inferred to be at least 50% and 23/28 reached this threshold on at least one target site. In contrast, only a single line (HD_CFD00032) resulted in no detectable gene editing at either sgRNA target site. This suggests that HD_CFD sgRNA lines mediate robust and efficient mutagenesis of target
Abstract Iron Regulatory Protein 1 (IRP1) is a bifunctional cytosolic iron sensor. When iron levels are normal, IRP1 harbours an iron-sulphur cluster (holo-IRP1), an enzyme with aconitase activity. When iron levels fall, IRP1 loses the cluster (apo-IRP1) and binds to iron-responsive elements (IREs) in messenger RNAs (mRNAs) encoding proteins involved in cellular iron uptake, distribution, and storage. Here we show that mutations in the Drosophila 1,4-Alpha-Glucan Branching Enzyme ( AGBE ) gene cause porphyria. AGBE was hitherto only linked to glycogen metabolism and a fatal human disorder known as glycogen storage disease type IV. AGBE binds specifically to holo-IRP1 and to mitoNEET, a protein capable of repairing IRP1 iron-sulphur clusters. This interaction ensures nuclear translocation of holo-IRP1 and downregulation of iron-dependent processes, demonstrating that holo-IRP1 functions not just as an aconitase, but throttles target gene expression in anticipation of declining iron requirements.
Abstract Intravenous injection provides a direct, rapid, and efficient route for delivering drugs or other substances, particularly for compounds with poor intestinal absorption or molecules (e.g. proteins) that are prone to structural changes and degradation within the digestive system. While Drosophila larvae represent a well-established genetic model for studying developmental and physiological pathways, as well as human diseases, their use in analyzing the molecular effects of substance exposure remains limited. In this study, we present a highly efficient injection method for Drosophila first- and second-instar larvae. Despite causing a slight developmental delay, this method achieves a high survival rate and offers a quick, easily adjustable protocol. The process requires 3–5 h to inject 150–300 larvae, depending on the microcapillary needle, microinjection system, and the compound being administered. As proof of concept, we compared the effects of injecting ferritin protein into Fer1HCH00451 mutant first instar larvae with those of dietary ferritin administration. Our results show that ferritin injection rescues Fer1HCH mutants, a result that cannot be achieved through dietary delivery. This approach is particularly valuable for the delivery of complex compounds in cases where oral administration is impaired or limited by the digestive system.
Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi , an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor ( EcR ) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.
Steroid hormones are powerful endocrine regulators, but little is known about how environmental conditions modulate steroidogenesis to reprogram developmental fates. Here, we use the Drosophila prothoracic gland (PG) to investigate how a nutrient restriction checkpoint (NRC) ensures or blocks developmental progression and sexual maturation via regulating steroidogenesis. Extensive transcriptome analysis of the PG reveals that pre-NRC starvation significantly downregulates mitochondria-associated genes. Pre-NRC starvation reduces prothoracicotropic neuropeptide hormone signaling, insulin signaling, and TORC1 activity in PG cells, which prevent mitochondrial fragmentation and import of Disembodied, a key steroidogenic enzyme. Ultimately, pre-NRC starvation causes severe mitophagy and proteasome dysfunction, blocking steroidogenesis and metamorphosis. By contrast, post-NRC starvation does not impair mitochondrial homeostasis in PG cells but reduces sit expression and induces moderate autophagy to promote steroidogenesis, leading to precocious metamorphosis. This study constitutes a paradigm for exploring how steroid hormone levels are controlled in response to environmental stress during developmental checkpoints. The prothoracic gland is the principal steroidogenic gland in Drosophila. Here Zhang et al. perform transcriptomic analysis and show that starvation prior to the nutrient restriction checkpoint alters mitochondrial homeostasis in the gland and blocks steroid hormone production.
The prothoracic gland (PG) is a major insect endocrine organ. It is the principal source of insect steroid hormones, and critical for key developmental events such as the molts, the establishment of critical weight (CW), pupation, and sexual maturation. However, little is known about the developmental processes that regulate PG morphology. In this study, we identified
Drosophila development is coordinated by pulses of the steroid hormone 20-hydroxyecdysone (20E). During metamorphosis, the 20E-inducible Broad-Complex (BR-C) gene plays a key role in the genetic hierarchies that transduce the hormone signal, being required for the destruction of larval tissues and numerous aspects of adult development. Most of the known BR-C target genes, including the salivary gland secretion protein (Sgs) genes, are terminal differentiation genes that are thought to be directly regulated by BR-C-encoded transcription factors. Here, we show that repression of Sgs expression is indirectly controlled by the BR-C through transcriptional down-regulation of fork head, a tissue-specific gene that plays a central role in salivary gland development and is required for Sgs expression. Our results demonstrate that integration of a tissue-specific regulatory gene into a 20E-controlled genetic hierarchy provides a mechanism for hormonal repression. Furthermore, they suggest that the BR-C is placed at a different position within the 20E-controlled hierarchies than previously assumed, and that at least part of its pleiotropic functions are mediated by tissue-specific regulators.
