Research report

2023
Nisha Singh, Agustin Rolandelli, Anya J O’Neal, Rainer L. Butler, Sourabh Samaddar, Hanna J Laukaitis-Yousey, Matthew Butnaru, Stephanie E Mohr, Norbert Perrimon, and Joao HF Pedra. 9/8/2023. “Genetic manipulation of an Ixodes scapularis cell line.” bioRxiv, Pp. 9/8/2023. 09.08.556855. Publisher's VersionAbstract
Although genetic manipulation is one of the hallmarks in 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 blacklegged tick Ixodes scapularis, an arthropod that serves as a vector of 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 alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticks in vivo and aid in circumventing the long-life cycle of I. scapularis, of which limits efficient and scalable molecular genetic screens.Importance Genetic 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 I. 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 A. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tick Ixodes scapularis.Competing Interest StatementThe authors have declared no competing interest.
2023.09.08.556855v1.full_.pdf
Nikki Coleman-Gosser, Yanhui Hu, Shiva Raghuvanshi, Shane Stitzinger, Weihang Chen, Arthur Luhur, Daniel Mariyappa, Molly Josifov, Andrew Zelhof, Stephanie E Mohr, Norbert Perrimon, and Amanda Simcox. 2023. “Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for research.” Elife, 12.Abstract

Expression of activated Ras, Ras, provides cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted Ras 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 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 Genomic Resource Center.

elife-85814-v2.pdf
Hong-Wen Tang, Kerstin Spirohn, Yanhui Hu, Tong Hao, István A Kovács, Yue Gao, Richard Binari, Donghui Yang-Zhou, Kenneth H Wan, Joel S Bader, Dawit Balcha, Wenting Bian, Benjamin W Booth, Atina G Coté, Steffi de Rouck, Alice Desbuleux, Kah Yong Goh, Dae-Kyum Kim, Jennifer J Knapp, Wen Xing Lee, Irma Lemmens, Cathleen Li, Mian Li, Roujia Li, Hyobin Julianne Lim, Yifang Liu, Katja Luck, Dylan Markey, Carl Pollis, Sudharshan Rangarajan, Jonathan Rodiger, Sadie Schlabach, Yun Shen, Dayag Sheykhkarimli, Bridget TeeKing, Frederick P. Roth, Jan Tavernier, Michael A Calderwood, David E Hill, Susan E Celniker, Marc Vidal, Norbert Perrimon, and Stephanie E. Mohr. 2023. “Next-generation large-scale binary protein interaction network for Drosophila melanogaster,” 14, 1, Pp. 2162. Publisher's VersionAbstract
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.
s41467-023-37876-0.pdf
Baolong Xia, Raghuvir Viswanatha, Yanhui Hu, Stephanie E Mohr, and Norbert Perrimon. 2023. “Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in cells.” Elife, 12.Abstract

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In 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 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 (), a member of the lipocalin protein family (), and a zinc finger C2H2 transcription factor (). Mechanistically, we demonstrate that overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in cells.

elife-85542-v1.pdf
Agustin Rolandelli, Hanna J Laukaitis-Yousey, Haikel N Bogale, Nisha Singh, Sourabh Samaddar, Anya J O’Neal, Camila R Ferraz, Matthew Butnaru, Enzo Mameli, Baolong Xia, Tays M. Mendes, Rainer L. Butler, Liron Marnin, Francy ECabrera Paz, Luisa M Valencia, Vipin S Rana, Ciaran Skerry, Utpal Pal, Stephanie E Mohr, Norbert Perrimon, David Serre, and Joao HF Pedra. 2023. “Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness.” bioRxiv, Pp. 2023.08.31.555785. Publisher's VersionAbstract
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 describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.Competing Interest StatementThe authors have declared no competing interest.
2023.08.31.555785v1.full_.pdf
2022
Shue Chen, Leah F Rosin, Gianluca Pegoraro, Nellie Moshkovich, Patrick J Murphy, Guoyun Yu, and Elissa P Lei. 8/12/2022. “NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization.” Nucleic Acids Res, 50, 14, Pp. 7906-7924.Abstract
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
gkac600.pdf
Ying Xu, Raghuvir Viswanatha, Oleg Sitsel, Daniel Roderer, Haifang Zhao, Christopher Ashwood, Cecilia Voelcker, Songhai Tian, Stefan Raunser, Norbert Perrimon, and Min Dong. 2022. “CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.” Nature, 610, 7931, Pp. 349-355.Abstract
Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Hans M Dalton, Raghuvir Viswanatha, Roderick Brathwaite, Jae Sophia Zuno, Alexys R Berman, Rebekah Rushforth, Stephanie E Mohr, Norbert Perrimon, and Clement Y Chow. 2022. “A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.” PLoS Genet, 18, 9, Pp. e1010430.Abstract
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.
Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Wei-Chun Tang, Chieh-Han Lu, Stephanie E. Mohr, Robert V. Farese, and Tobias C. Walther. 2022. “Identification of two pathways mediating protein targeting from ER to lipid droplets.” Nature Cell Biol. Publisher's VersionAbstract
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.
s41556-022-00974-0-1.pdf
Jun Xu, Ah-Ram Kim, Ross W Cheloha, Fabian A Fischer, Joshua Shing Shun Li, Yuan Feng, Emily Stoneburner, Richard Binari, Stephanie E Mohr, Jonathan Zirin, Hidde L Ploegh, and Norbert Perrimon. 2022. “Protein visualization and manipulation in through the use of epitope tags recognized by nanobodies.” Elife, 11.Abstract
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 (<15 aa) 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.
2021
Hans M. Dalton, Raghuvir Viswanatha, Ricky Brathwaite Jr., Jae Sophia Zuno, Stephanie E Mohr, Norbert Perrimon, and Clement Y. Chow. 12/4/2021. “A genome-wide CRISPR screen identifies the glycosylation enzyme DPM1 as a modifier of DPAGT1 deficiency and ER stress.” BioRxiv. Publisher's VersionAbstract
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 cause CDGs. While both in vivo models ostensibly cause ER 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.
2021.12.03.471178v1.full_.pdf
Raghuvir Viswanatha, Enzo Mameli, Jonathan Rodiger, Pierre Merckaert, Fabiana Feitosa-Suntheimer, Tonya M Colpitts, Stephanie E Mohr, Yanhui Hu, and Norbert Perrimon. 11/24/2021. “Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos.” Nat Commun, 12, 1, Pp. 6825.Abstract
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.
s41467-021-27129-31.pdf
Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Chieh-Han Lu, Stephanie E. Mohr, Jr Robert V. Farese, and Tobias C. Walther. 9/15/2021. “Identification of two pathways mediating protein targeting from ER to lipid droplets [NOTE: a modified final version was published in Nat Cell Biol and is now available]”. Publisher's VersionAbstract
Pathways localizing proteins to their sites of action within a cell are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, little is known about how proteins, such as key metabolic enzymes, target from the ER to cellular lipid droplets (LDs). Here, we identify two distinct pathways for ER-to-LD (ERTOLD) protein targeting: early ERTOLD, occurring during LD formation, and late ERTOLD, targeting mature LDs after their formation. By using systematic, unbiased approaches, we identified specific membrane-fusion machinery, including regulators, a tether, and SNARE proteins, that are required for late ERTOLD targeting. Components of this fusion machinery localize to LD-ER interfaces and appear to be organized at ER exit sites (ERES) to generate ER-LD membrane bridges. We also identified multiple cargoes for early and late ERTOLD. Collectively, our data provide a new model for how proteins target LDs from the ER.
2021.09.14.460330v1.full_.pdf
Raghuvir Viswanatha, Enzo Mameli, Jonathan Rodiger, Pierre Merckaert, Fabiana Feitosa-Suntheimer, Tonya M. Colpitts, Stephanie E. Mohr, Yanhui Hu, and Norbert Perrimon. 3/30/2021. “Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos [NOTE: A modified final version was published in Nat Comm and is now available.].” bioRxiv. Publisher's VersionAbstract
Mosquito-borne diseases present a worldwide public health burden. Genome-scale screening tools that could inform our understanding of mosquitos and their control are lacking. Here, we adapt a recombination-mediated cassette exchange system for delivery of CRISPR sgRNA libraries into cell lines from several mosquito species and perform pooled CRISPR screens in an Anopheles cell line. To implement this method, we engineered modified mosquito cell lines, validated promoters and developed bioinformatics tools for multiple mosquito species.Competing Interest StatementThe authors have declared no competing interest.
2021.03.29.437496v2.full_.pdf
Xiangzhao Yue, Yongkang Liang, Zhishuang Wei, Jun Lv, Yongjin Cai, Xiaobin Fan, Wenqing Zhang, and Jie Chen. 2021. “Genome-wide in vitro and in vivo RNAi screens reveal Fer3 to be an important regulator of kkv transcription in Drosophila.” Insect Sci.Abstract
Krotzkopf verkehrt (kkv) is a key enzyme that catalyzes the synthesis of chitin, an important component of the Drosophila epidermis, trachea, and other tissues. Here, we report the use of comprehensive RNA interference (RNAi) analyses to search for kkv transcriptional regulators. A cell-based RNAi screen identified 537 candidate kkv regulators on a genome-wide scale. Subsequent use of transgenic Drosophila lines expressing RNAi constructs enabled in vivo validation, and we identified six genes as potential kkv transcriptional regulators. Weakening of the kkvDsRed signal, an in vivo reporter indicating kkv promoter activity, was observed when the expression of Akirin, NFAT, 48 related 3 (Fer3), or Autophagy-related 101(Atg101) was knocked down in Drosophila at the 3rd-instar larval stage; whereas we observed disoriented taenidial folds on larval tracheae when Lines (lin) or Autophagy-related 3(Atg3) was knocked down in the tracheae. Fer3, in particular, has been shown to be an important factor in the activation of kkv transcription via specific binding with the kkv promoter. The genes involved in the chitin synthesis pathway were widely affected by the downregulation of Fer3. Furthermore, Atg101, Atg3, Akirin, Lin, NFAT, Pnr and Abd-A showed the potential complex mechanism of kkv transcription are regulated by an interaction network with bithorax complex components. Our study revealed the hitherto unappreciated diversity of modulators impinging on kkv transcription and opens new avenues in the study of kkv regulation and chitin biosynthesis. This article is protected by copyright. All rights reserved.
Xuechun Feng, Víctor López Del Amo, Enzo Mameli, Megan Lee, Alena L Bishop, Norbert Perrimon, and Valentino M Gantz. 2021. “Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes.” Nat Commun, 12, 1, Pp. 2960.Abstract
Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species.
s41467-021-23239-0.pdf
J. A. Bosch, G. Birchak, and N. Perrimon. 2021. “Precise genome engineering in Drosophila using prime editing.” Proc Natl Acad Sci U S A, 118.Abstract
Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.
2020
R. Viswanatha, M. Zaffagni, J. Zirin, N. Perrimon, and S. Kadener. 11/1/2020. “CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells.” BioRxiv.Abstract
Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.
2020.11.01.364166v1.full_.pdf
Jonathan Zirin, Yanhui Hu, Luping Liu, Donghui Yang-Zhou, Ryan Colbeth, Dong Yan, Ben Ewen-Campen, Rong Tao, Eric Vogt, Sara VanNest, Cooper Cavers, Christians Villalta, Aram Comjean, Jin Sun, Xia Wang, Yu Jia, Ruibao Zhu, Ping Peng, Jinchao Yu, Da Shen, Yuhao Qiu, Limmond Ayisi, Henna Ragoowansi, Ethan Fenton, Senait Efrem, Annette Parks, Kuniaki Saito, Shu Kondo, Liz Perkins, Stephanie E Mohr, Jianquan Ni, and Norbert Perrimon. 2020. “Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies.” Genetics.Abstract
The Transgenic RNAi Project (TRiP), a functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNAi fly stocks. To date, the TRiP has generated >15,000 RNAi fly stocks. As this covers most genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express sgRNAs targeting upstream of a gene transcription start site. Gene activation is triggered by co-expression of catalytically dead Cas9 (dCas9) fused to an activator domain, either VP64-p65-Rta (VPR) or Synergistic Activation Mediator (SAM). TRiP-KO stocks express one or two sgRNAs targeting the coding sequence of a gene or genes. Cutting is triggered by co-expression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated more than 5,000 CRISPR-OE or -KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.
2019
Justin A. Bosch, Ryan Colbeth, Jonathan Zirin, and Norbert Perrimon. 5/16/2019. “Gene knock-ins in Drosophila using homology-independent insertion of universal donor plasmids.” BioRxiv. Publisher's Version 2019_BioRxiv_Bosch .pdf

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