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The design, analysis and mining of large-scale 'omics studies with the goal of advancing biological and biomedical understanding require use of a range of bioinformatics tools, including approaches tailored to needs specific to a given species and/or technology. The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) Functional Genomics Resources (https://fgr.hms.harvard.edu/tools) supports an increasingly broad group of technologies and species. Recently, for example, we expanded the database to include additional new data-centric resources that facilitate mining and analysis of single-cell transcriptomics. In addition, we have applied our approaches to CRISPR reagent and gene-centric bioinformatics approaches in Drosophila to arthropod vectors of infectious diseases. Building on our previous comprehensive reports on the FlyRNAi database, here we focus on new and updated resources with a primary focus on data-centric tools. Altogether, our suite of online resources supports various stages of functional genomics studies for Drosophila and other arthropods, and facilitate a wide range of reagent design, analysis, data mining and analysis approaches by biologists and biomedical experts studying Drosophila, other common genetic model species, arthropod vectors and/or human biology., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a pooled CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries, followed by PCR and sequencing of integrated sgRNAs. While effective, the method relies on early constitutive Cas9 activity that potentially can lead to discrepancies between genome edits and sgRNAs detected by PCR, reducing screen accuracy. To address this issue, we introduce a new method to co-transfect a plasmid expressing the anti-CRISPR protein AcrIIa4 to suppress Cas9 activity during early sgRNA expression, which we term "IntAC" (integrase with anti-CRISPR). IntAC allowed us to construct a new CRISPR screening approach driven by the high strength dU6:3 promoter. This new library dramatically improved precision-recall of fitness genes across the genome, retrieving 90-95% of essential gene groups within 5% error, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila . Our analysis determined that elevated sgRNA levels, made permissible by the IntAC approach, drove much of the improvement. The Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We further demonstrate that IntAC combined with a targeted sgRNA sub-library enabled precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell types, including mosquito, lepidopteran, tick, and mammalian cells.

Anopheles mosquitoes are the sole vector of human malaria, the most burdensome vector-borne disease worldwide. Strategies aimed at reducing mosquito populations and limiting their ability to transmit disease show the most promise for disease control. Therefore, gaining an improved understanding of mosquito biology, and specifically that of the immune response, can aid efforts to develop new approaches that limit malaria transmission. Here, we use a genome-wide CRISPR screening approach for the first time in mosquito cells to identify essential genes in Anopheles and identify genes for which knockout confers resistance to clodronate liposomes, which have been widely used in mammals and arthropods to ablate immune cells. In the essential gene screen, we identified a set of 1280 Anopheles genes that are highly enriched for genes involved in fundamental cell processes. For the clodronate liposome screen, we identified several candidate resistance factors and confirm their roles in the uptake and processing of clodronate liposomes through in vivo validation in Anopheles gambiae , providing new mechanistic detail of phagolysosome formation and clodronate liposome function. In summary, we demonstrate the application of a genome-wide CRISPR knockout platform in a major malaria vector and the identification of genes that are important for fitness and immune-related processes.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis ( xiap ) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks , which currently limit efficient and scalable molecular genetic screens in vivo .IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47 , two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis . Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum . Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis ., Competing Interests: The authors declare no conflict of interest.

Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods., (© 2024. The Author(s).)

Genes that have been identified in the genome but remain uncharacterized with regards to function offer an opportunity to uncover novel biological information. Novelty is exciting but can also be a barrier. If nothing is known, how does one start planning and executing experiments? Here, we provide a recommended information-mining workflow and a corresponding guide to accessing information about uncharacterized Drosophila melanogaster genes, such as those assigned only a systematic coding gene identifier. The available information can provide insights into where and when the gene is expressed, what the function of the gene might be, whether there are similar genes in other species, whether there are known relationships to other genes, and whether any other features have already been determined. In addition, available information about relevant reagents can inspire and facilitate experimental studies. Altogether, mining available information can help prioritize genes for further study, as well as provide starting points for experimental assays and other analyses., Competing Interests: Conflicts of interest statement The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Expression of activated Ras, Ras V12 , provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted Ras V12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad Ras V12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center., Competing Interests: NC, YH, SR, SS, WC, AL, DM, MJ, AZ, SM, NP, AS No competing interests declared

Gene set enrichment analysis (GSEA) plays an important role in large-scale data analysis, helping scientists discover the underlying biological patterns over-represented in a gene list resulting from, for example, an 'omics' study. Gene Ontology (GO) annotation is the most frequently used classification mechanism for gene set definition. Here we present a new GSEA tool, PANGEA (PAthway, Network and Gene-set Enrichment Analysis; https://www.flyrnai.org/tools/pangea/), developed to allow a more flexible and configurable approach to data analysis using a variety of classification sets. PANGEA allows GO analysis to be performed on different sets of GO annotations, for example excluding high-throughput studies. Beyond GO, gene sets for pathway annotation and protein complex data from various resources as well as expression and disease annotation from the Alliance of Genome Resources (Alliance). In addition, visualizations of results are enhanced by providing an option to view network of gene set to gene relationships. The tool also allows comparison of multiple input gene lists and accompanying visualisation tools for quick and easy comparison. This new tool will facilitate GSEA for Drosophila and other major model organisms based on high-quality annotated information available for these species., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters ( CG8468 ), a member of the lipocalin protein family ( CG5399 ), and a zinc finger C2H2 transcription factor ( CG9932 ). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells., Competing Interests: BX, RV, YH, SM, NP No competing interests declared, (© 2023, Xia et al.)

Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function., (© 2023. The Author(s).)

Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Nanobody production in S2 cells Basic Protocol 2: Nanobody expression and purification in bacterial cells Basic Protocol 3: Immunostaining with nanobodies Basic Protocol 4: Immunoblotting with nanobodies Basic Protocol 5: Immunoprecipitation with nanobodies prepared from S2 cells Basic Protocol 6: Immunoprecipitation with nanobodies prepared from bacteria Basic Protocol 7: NbVHH05 and Nb127D01 used as chromobodies Basic Protocol 8: NanoTag trap as a method to alter protein localization Support Protocol: CRISPR-mediated tagging of endogenous genes with NanoTags., (© 2022 Wiley Periodicals LLC.)

Paralogs are genes which arose via gene duplication, and when such paralogs retain overlapping or redundant function, this poses a challenge to functional genetics research. Recent technological advancements have made it possible to systematically probe gene function for redundant genes using dual or multiplex gene perturbation, and there is a need for a simple bioinformatic tool to identify putative paralogs of a gene(s) of interest. We have developed Paralog Explorer (https://www.flyrnai.org/tools/paralogs/), an online resource that allows researchers to quickly and accurately identify candidate paralogous genes in the genomes of the model organisms D. melanogaster, C. elegans, D. rerio, M. musculus, and H. sapiens . Paralog Explorer deploys an effective between-species ortholog prediction software, DIOPT, to analyze within-species paralogs. Paralog Explorer allows users to identify candidate paralogs, and to navigate relevant databases regarding gene co-expression, protein-protein and genetic interaction, as well as gene ontology and phenotype annotations. Altogether, this tool extends the value of current ortholog prediction resources by providing sophisticated features useful for identification and study of paralogous genes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress., Competing Interests: The authors have declared that no competing interests exist.

Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD-ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges., (© 2022. The Author(s).)

For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond., Competing Interests: JX, AK, RC, FF, JL, YF, ES, RB, SM, JZ, HP, NP No competing interests declared, (© 2022, Xu et al.)

Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species., (© 2021. The Author(s).)