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DRSC-BTRR Driving Biomedical Projects

The goal of DRSC-BTRR Driving Biomedical Projects (DBPs) is to partner our technology development efforts with unmet needs in the community. Collabortors engage with us in the technology development process and provide critical feedback, feeding an iterative cycle of technology development and improvement. Please feel free to contact the DRSC-BTRR Director S.E. Mohr to discuss potential new collaborations (contact link on the DRSC-BTRR home page).

Interested to use our technologies to help address your biomedical topic of interest? Contact DRSC-BTRR Director Stephanie Mohr.

In addition to DBPs, we also engage in more focused Collaboration and Service Projects including by providing reagents for fly cell RNAi studies, and regularly provide advice on use of RNAi and CRISPR technologies in flies and fly cells.

The DRSC-BTRR is engaged in the following DBPs
(last udpated May 2023)

In progress:

M. Bulyk, Brigham & Women's Hospital, Use of the NanoTag system for ChIPseq and related studies

S. Cherry, University of Pennsylvania, CRISPR knockout screens in Aedes mosquito cells to identify host factors related to viral entry

F. Douam, Boston University and NEIDL, Establishing assays relevant to infection of mosquito cells by viral pathogens, for mosquito CRISPR pooled cell screening

S. Kadener, Brandeis University, Adapting the RNA-targeting Cas13 system for use in vivo in Drosophila to study circular RNAs. Preliminary resulted reported in BioRxiv (Viswanatha et al. 2020)

W. Kaelin, Dana Farber Cancer Institute, Cross-species, functional genomics screening to identify synthetic lethal interactors with tumor suppressor genes.

R. Lehmann, Whitehead Institute/MIT, Identification of protein binders for the study of Wolbachia in Drosophila.

R. Smith, Iowa State University, Pooled CRISPR screening in Anopheles cell lines to investigate cell biological activities.

 

Recently completed:

H. Bellen, Baylor College of Medicine, Use of Drosophila cultured cells as a test system for optimization of new MiMIC system CRISPR knock-in approaches and cassettes. Published. See links below.

C. Chow, University of Utah, Drosophila CRISPR pooled cell screens to explore cell biological topics related to rare disorders of glycosylation. Published. See links below.

T. Colpitts, Boston University & National Emerging Infectious Diseases Laboratory (NEIDL), Establishing assays relevant to infection of mosquito cells by viral pathogens, for mosquito CRISPR pooled cell screening. Published. See links below.

M. Dong, Boston Children’s Hospital, Genome-wide CRISPR/Cas9 screen to identify the receptors for insecticidal toxins. Published. See links below.

V. Gantz, UCSD, Use of Culex cell lines as a testing platform for CRISPR-Cas9 optimization for eventual use in gene drive applications in vivo. Published. See links below.

Preprints and publications resulting from DRSC-BTRR DBPs

  • 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, 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
    2021.12.03.471178v1.full_.pdf
    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.
  • O. Kanca, J. Zirin, Y Hu, B. Tepe, D. Dutta, W. W Lin, L. Ma, M Ge, Z. Zuo, L. P Liu, R. W Levis, N. Perrimon, and H. J Bellen. Published. “An expanded toolkit for Drosophila gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination.” Elife 11.Abstract
    Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate.
  • 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
    2021.03.29.437496v2.full_.pdf
    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.
  • Thomas A Ravenscroft, Jennifer B Phillips, Elizabeth Fieg, Sameer S Bajikar, Judy Peirce, Jeremy Wegner, Alia A Luna, Eric J Fox, Yi-Lin Yan, Jill A Rosenfeld, Jonathan Zirin, Oguz Kanca, Undiagnosed Diseases Network, Paul J Benke, Eric S Cameron, Vincent Strehlow, Konrad Platzer, Rami Abou Jamra, Chiara Klöckner, Matthew Osmond, Thomas Licata, Samantha Rojas, David Dyment, Josephine SC Chong, Sharyn Lincoln, Joan M Stoler, John H Postlethwait, Michael F Wangler, Shinya Yamamoto, Joel Krier, Monte Westerfield, and Hugo J Bellen. 2021. “Heterozygous loss-of-function variants significantly expand the phenotypes associated with loss of GDF11.” Genet Med, 23, 10, Pp. 1889-1900.Abstract
    2022_genetics_in_medicine_ravenscroft.pdf
    2022_genetics_in_medicine_ravenscroft_supplementals.zip
    PURPOSE: Growth differentiation factor 11 (GDF11) is a key signaling protein required for proper development of many organ systems. Only one prior study has associated an inherited GDF11 variant with a dominant human disease in a family with variable craniofacial and vertebral abnormalities. Here, we expand the phenotypic spectrum associated with GDF11 variants and document the nature of the variants. METHODS: We present a cohort of six probands with de novo and inherited nonsense/frameshift (4/6 patients) and missense (2/6) variants in GDF11. We generated gdf11 mutant zebrafish to model loss of gdf11 phenotypes and used an overexpression screen in Drosophila to test variant functionality. RESULTS: Patients with variants in GDF11 presented with craniofacial (5/6), vertebral (5/6), neurological (6/6), visual (4/6), cardiac (3/6), auditory (3/6), and connective tissue abnormalities (3/6). gdf11 mutant zebrafish show craniofacial abnormalities and body segmentation defects that match some patient phenotypes. Expression of the patients' variants in the fly showed that one nonsense variant in GDF11 is a severe loss-of-function (LOF) allele whereas the missense variants in our cohort are partial LOF variants. CONCLUSION: GDF11 is needed for human development, particularly neuronal development, and LOF GDF11 alleles can affect the development of numerous organs and tissues.
  • 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
    2020.11.01.364166v1.full_.pdf
    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.
  • 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.
  • 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
    s41467-021-23239-0.pdf
    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.