The pleiotropic functions of Pri smORF peptides synchronize leg development regulators.

The last decade witnesses the emergence of the abundant family of smORF peptides, encoded by small ORF (<100 codons), whose biological functions remain largely unexplored. Bioinformatic analyses here identify hundreds of putative smORF peptides expressed in Drosophila imaginal leg discs. Thanks to a functional screen in leg, we found smORF peptides involved in morphogenesis, including the pioneer smORF peptides Pri. Since we identified its target Ubr3 in the epidermis and pri was known to control leg development through poorly understood mechanisms, we investigated the role of Ubr3 in mediating pri function in leg. We found that pri plays several roles during leg development both in patterning and in cell survival. During larval stage, pri activates independently of Ubr3 tarsal transcriptional programs and Notch and EGFR signaling pathways, whereas at larval pupal transition, Pri peptides cooperate with Ubr3 to insure cell survival and leg morphogenesis. Our results highlight Ubr3 dependent and independent functions of Pri peptides and their pleiotropy. Moreover, we reveal that the smORF peptide family is a reservoir of overlooked developmental regulators, displaying distinct molecular functions and orchestrating leg development.

A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression.

The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors.

A temperature-tolerant CRISPR base editor mediates highly efficient and precise gene editing in Drosophila.

CRISPR nucleases generate a broad spectrum of mutations that includes undesired editing outcomes. Here, we develop optimized C-to-T base editing systems for the generation of precise loss- or gain-of-function alleles in Drosophila and identify temperature as a crucial parameter for efficiency. We find that a variant of the widely used APOBEC1 deaminase has attenuated activity at 18° to 29°C and shows considerable dose-dependent toxicity. In contrast, the temperature-tolerant evoCDA1 domain mediates editing of typically more than 90% of alleles and is substantially better tolerated. Furthermore, formation of undesired mutations is exceptionally rare in Drosophila compared to other species. The predictable editing outcome, high efficiency, and product purity enables near homogeneous induction of STOP codons or alleles encoding protein variants in vivo. Last, we demonstrate how optimized expression enables conditional base editing in marked cell populations. This work substantially facilitates creation of precise alleles in Drosophila and provides key design parameters for developing efficient base editing systems in other ectothermic species.

A global genetic interaction network by single-cell imaging and machine learning.

Cellular and organismal phenotypes are controlled by complex gene regulatory networks. However, reference maps of gene function are still scarce across different organisms. Here, we generated synthetic genetic interaction and cell morphology profiles of more than 6,800 genes in cultured Drosophila cells. The resulting map of genetic interactions was used for machine learning-based gene function discovery, assigning functions to genes in 47 modules. Furthermore, we devised Cytoclass as a method to dissect genetic interactions for discrete cell states at the single-cell resolution. This approach identified an interaction of Cdk2 and the Cop9 signalosome complex, triggering senescence-associated secretory phenotypes and immunogenic conversion in hemocytic cells. Together, our data constitute a genome-scale resource of functional gene profiles to uncover the mechanisms underlying genetic interactions and their plasticity at the single-cell level.

USP47 deubiquitylates Groucho/TLE to promote Wnt-ß-catenin signaling.

The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.

Tissue-Specific CRISPR-Cas9 Screening in Drosophila.

Over the last century research in Drosophila has resulted in many fundamental contributions to our understanding of the biology of multicellular organisms. Many of these breakthroughs have been based on the identification of novel gene functions in large-scale genetic screens. However, conventional forward-genetic screens have been limited by the random nature of mutagenesis and difficulties in mapping causal mutations, while reverse-genetic RNAi screens suffer from incomplete knockdown of gene expression. Recently developed large-scale CRISPR-Cas9 libraries promise to address these limitations by allowing the induction of targeted mutations in genes with spatial and temporal control. Here, we provide a guide for tissue-specific CRISPR screening in Drosophila, including the characterization of Gal4 UAS-Cas9 lines, selection of sgRNA libraries, and various quality control measures. We also discuss confounding factors that can give rise to false-positive and false-negative results in such experiments and suggest strategies on how to detect and avoid them. Conditional CRISPR screening represents an exciting new approach for functional genomics in vivo and is set to further expand our knowledge of the molecular underpinning of development, homeostasis, and disease.

Conditional CRISPR-Cas Genome Editing in Drosophila to Generate Intestinal Tumors.

CRISPR-Cas has revolutionized genetics and extensive efforts have been made to enhance its editing efficiency by developing increasingly more elaborate tools. Here, we evaluate the CRISPR-Cas9 system in Drosophila melanogaster to assess its ability to induce stem cell-derived tumors in the intestine. We generated conditional tissue-specific CRISPR knockouts using different Cas9 expression vectors with guide RNAs targeting the BMP, Notch, and JNK pathways in intestinal progenitors such as stem cells (ISCs) and enteroblasts (EBs). Perturbing Notch and BMP signaling increased the proliferation of ISCs/EBs and resulted in the formation of intestinal tumors, albeit with different efficiencies. By assessing both the anterior and posterior regions of the midgut, we observed regional differences in ISC/EB proliferation and tumor formation upon mutagenesis. Surprisingly, high continuous expression of Cas9 in ISCs/EBs blocked age-dependent increase in ISCs/EBs proliferation and when combined with gRNAs targeting tumor suppressors, it prevented tumorigenesis. However, no such effects were seen when temporal parameters of Cas9 were adjusted to regulate its expression levels or with a genetically modified version, which expresses Cas9 at lower levels, suggesting that fine-tuning Cas9 expression is essential to avoid deleterious effects. Our findings suggest that modifications to Cas9 expression results in differences in editing efficiency and careful considerations are required when choosing reagents for CRISPR-Cas9 mutagenesis studies. In summary, Drosophila can serve as a powerful model for context-dependent CRISPR-Cas based perturbations and to test genome-editing systems in vivo.

Multiplexed conditional genome editing with Cas12a in Drosophila.

CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.

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 (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.

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.

Robust Wnt signaling is maintained by a Wg protein gradient and Fz2 receptor activity in the developing Drosophila wing.

Wnts are secreted proteins that regulate cell fate during development of all metazoans. Wnt proteins were proposed to spread over several cells to activate signaling directly at a distance. In the Drosophila wing epithelium, an extracellular gradient of the Wnt1 homolog Wingless (Wg) was observed extending over several cells away from producing cells. Surprisingly, however, it was also shown that a membrane-tethered Neurotactin-Wg fusion protein (NRT-Wg) can largely replace endogenous Wg, leading to proper patterning of the wing. Therefore, the functional range of Wg and whether Wg spreading is required for correct tissue patterning remains controversial. Here, by capturing secreted Wg on cells away from the source, we show that Wg acts over a distance of up to 11 cell diameters to induce signaling. Furthermore, cells located outside the reach of extracellular Wg depend on the Frizzled2 receptor to maintain signaling. Frizzled2 expression is increased in the absence of Wg secretion and is required to maintain signaling and cell survival in NRT-wg wing discs. Together, these results provide insight into the mechanisms by which robust Wnt signaling is achieved in proliferating tissues.

RNA Interference (RNAi) Screening in Drosophila.

In the last decade, RNA interference (RNAi), a cellular mechanism that uses RNA-guided degradation of messenger RNA transcripts, has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. We will discuss available reagent libraries and cell lines, methodological approaches for cell-based assays, and computational methods for the analysis of high-throughput screens. Furthermore, we will review the generation and use of genome-scale RNAi libraries for tissue-specific knockdown analysis in vivo and discuss the differences and similarities with the use of genome-engineering methods such as CRISPR/Cas9 for functional analysis.

Creating Heritable Mutations in Drosophila with CRISPR-Cas9.

Reverse genetics-the creation of mutations in preselected target genes-has until recently been a bottleneck in many Drosophila projects. The advent of clustered, regularly interspaced, short palindromic repeat (CRISPR) genome engineering systems has transformed this situation. A short time after the in vitro demonstration of target site cleavage by the RNA-guided endonuclease CRISPR-associated nuclease 9 (Cas9) (Jinek et al., Science 337:816-821, 2012), hundreds of fly researchers are using CRISPR technology to generate loss-of-function mutant alleles in specific genes, as well as to create specific point mutations or tagged protein products. It appears that most target genes can be edited with remarkably high efficiency, with engineered strains often available a few weeks after conception of a project. Here, we provide a step-by-step protocol for creating loss-of-function mutations in Drosophila using transgenic Cas9 sources, which is based on optimized reagents and procedures that have been evaluated in our laboratory. We also provide guidance on extending this protocol to produce precise genomic alterations by homology-directed repair in the presence of a donor sequence. Additional information and updates are available from our website, www.crisprflydesign.org .

Augmenting CRISPR applications in Drosophila with tRNA-flanked sgRNAs.

We present tRNA-based vectors for producing multiple clustered regularly interspaced short palindromic repeats (CRISPR) single guide RNAs (sgRNAs) from a single RNA polymerase II or III transcript in Drosophila. The system, which is based on liberation of sgRNAs by processing flanking tRNAs, permits highly efficient multiplexing of Cas9-based mutagenesis. We also demonstrate that the tRNA-sgRNA system markedly increases the efficacy of conditional gene disruption by Cas9 and can promote editing by the recently discovered RNA-guided endonuclease Cpf1.

Aubergine iCLIP Reveals piRNA-Dependent Decay of mRNAs Involved in Germ Cell Development in the Early Embryo.

The Piwi-interacting RNA (piRNA) pathway plays an essential role in the repression of transposons in the germline. Other functions of piRNAs such as post-transcriptional regulation of mRNAs are now emerging. Here, we perform iCLIP with the PIWI protein Aubergine (Aub) and identify hundreds of maternal mRNAs interacting with Aub in the early Drosophila embryo. Gene expression profiling reveals that a proportion of these mRNAs undergo Aub-dependent destabilization during the maternal-to-zygotic transition. Strikingly, Aub-dependent unstable mRNAs encode germ cell determinants. iCLIP with an Aub mutant that is unable to bind piRNAs confirms piRNA-dependent binding of Aub to mRNAs. Base pairing between piRNAs and mRNAs can induce mRNA cleavage and decay that are essential for embryonic development. These results suggest general regulation of maternal mRNAs by Aub and piRNAs, which plays a key developmental role in the embryo through decay and localization of mRNAs encoding germ cell determinants.

Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research.

The Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated (CRISPR/Cas) technology allows rapid, site-specific genome modification in a wide variety of organisms . Proof-of-principle studies in Drosophila melanogaster have used various CRISPR/Cas tools and experimental designs, leading to significant uncertainty in the community about how to put this technology into practice. Moreover, it is unclear what proportion of genomic target sites can be modified with high efficiency. Here, we address these issues by systematically evaluating available CRISPR/Cas reagents and methods in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for somatic and germline genome engineering and conclude that optimized CRISPR with independent transgenes is as efficient, more versatile, and does not represent a biosafety risk.

Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.

The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system has emerged recently as a powerful method to manipulate the genomes of various organisms. Here, we report a toolbox for high-efficiency genome engineering of Drosophila melanogaster consisting of transgenic Cas9 lines and versatile guide RNA (gRNA) expression plasmids. Systematic evaluation reveals Cas9 lines with ubiquitous or germ-line-restricted patterns of activity. We also demonstrate differential activity of the same gRNA expressed from different U6 snRNA promoters, with the previously untested U6:3 promoter giving the most potent effect. An appropriate combination of Cas9 and gRNA allows targeting of essential and nonessential genes with transmission rates ranging from 25-100%. We also demonstrate that our optimized CRISPR/Cas tools can be used for offset nicking-based mutagenesis. Furthermore, in combination with oligonucleotide or long double-stranded donor templates, our reagents allow precise genome editing by homology-directed repair with rates that make selection markers unnecessary. Last, we demonstrate a novel application of CRISPR/Cas-mediated technology in revealing loss-of-function phenotypes in somatic cells following efficient biallelic targeting by Cas9 expressed in a ubiquitous or tissue-restricted manner. Our CRISPR/Cas tools will facilitate the rapid evaluation of mutant phenotypes of specific genes and the precise modification of the genome with single-nucleotide precision. Our results also pave the way for high-throughput genetic screening with CRISPR/Cas.

A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.

Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.

A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion.

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.

Wnt signalling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt-producing cells.

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.