Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR)

The NIH NIGMS P41-funded DRSC-BTRR helps researchers realize the full potential of Drosophila melanogaster as a model for the study of human health and disease.

The DRSC-BTRR develops state-of-the-art tools and methods in three technology areas: (1) Development of technologies for Drosophila cell-based and in vivo studies, (2) Application of technologies for study of mosquito vectors of human diseases, and (3) Development of in vivo proteomics technologies for Drosophila.

We develop technologies through iterative rounds of testing and improvement together with ‘driving biomedical projects’ at collaborating labs that can benefit from the technologies. Current collaborators include experts in cancer therapeutics, rare genetic diseases, and mosquito vectors of infectious diseases.

To further extend the impact of the technologies, we engage in community activities that inform a broad audience and rapidly disseminate technologies. Altogether, we will serve as an integrated, collaborative resource engaging in projects with strong potential for impact in areas that are of interest to several institutes at the US National Institutes of Health.

Point-of-contact for inquiries about DSRC-BTRR technologies and collaborations: Stephanie Mohr

Technology Research & Development (TR&D) focus areas:

TR&D1: Development of CRISPR-based functional genomics technologies for high-throughput screening in Drosophila cultured cells and for use in vivo in Drosophila. Read more about CRISPR knockout in Drosophila cells here.

TR&D2: Development of CRISPR-based functional genomics and other technologies for use in mosquitos, including development of CRISPR screening technologies for use in mosquito cell lines. Read more about mosquito cell technologies here.

TR&D3: Development of proteomics-based technologies for use in vivo in Drosophila, including development of new protein binding and labeling technologies.

Additional components of the DRSC-BTRR include

  • Driving Biomedical Projects (DBPs), which allow us to directly meet the needs of collaborators through iterative technology testing and development
  • Collaboration & Service Projects (CSPs), such as Drosophila cell-based RNAi screens using established technologies
  • Community Engagement, including presentations and workshops aimed at helping the broadest possible research community access DRSC-BTRR technologies
  • Administration & Management, including oversight by NIH NIGMS leadership and scientific advisors to the DRSC-BTRR
Diagram of the interaction between the DRSC-BTRR (tech development) and Driving Biomedical Projects (tech testing)
  • Drosophila melangaster 'fruit flies' with visible phenotypes, such as differences in the eye as compared with wild-type flies, due to mutation in 'marker' genes used in genetic analyses and transgenesis
Diagram of leadship roles at the DRSC-BTRR (Perrimon, PI; Mohr, Director: et al)

Recent Publications from the DSRC-BTRR

R. Viswanatha, M. Zaffagni, J. Zirin, N. Perrimon, and S. Kadener. Submitted. “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.
Y. Hu, S.G. Tattikota, Y. Liu, A. Comjean, Y. Gao, C. Forman, G. Kim, J. Rodiger, I. Papatheodorou, G. dos Santos, S.E. Mohr, and N. Perrimon. Submitted. “DRscDB: A single-cell RNA-seq resource for data mining and data comparison across species.” BioRxiv. Publisher's VersionAbstract
With the advent of single-cell RNA sequencing (scRNA-seq) technologies, there has been a spike in studies involving scRNA-seq of several tissues across diverse species including Drosophila. Although a few databases exist for users to query genes of interest within the scRNA-seq studies, search tools that enable users to find orthologous genes and their cell type-specific expression patterns across species are limited. Here, we built a new search database, called DRscDB (https://www.flyrnai.org/tools/single_cell/web/) to address this need. DRscDB serves as a comprehensive repository for published scRNA-seq datasets for Drosophila and the relevant datasets from human and other model organisms. DRscDB is based on manual curation of Drosophila scRNA-seq studies of various tissue types and their corresponding analogous tissues in vertebrates including zebrafish, mouse, and human. Of note, our search database provides most of the literature-derived marker genes, thus preserving the original analysis of the published scRNA-seq datasets. DRscDB serves as a web-based user interface that allows users to mine, utilize and compare gene expression data pertaining to scRNA-seq datasets from the published literature.
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.
Yanhui Hu, Verena Chung, Aram Comjean, Jonathan Rodiger, Fnu Nipun, Norbert Perrimon, and Stephanie E Mohr. 2020. “BioLitMine: Advanced Mining of Biomedical and Biological Literature About Human Genes and Genes from Major Model Organisms.” G3 (Bethesda).Abstract
The accumulation of biological and biomedical literature outpaces the ability of most researchers and clinicians to stay abreast of their own immediate fields, let alone a broader range of topics. Although available search tools support identification of relevant literature, finding relevant and key publications is not always straightforward. For example, important publications might be missed in searches with an official gene name due to gene synonyms. Moreover, ambiguity of gene names can result in retrieval of a large number of irrelevant publications. To address these issues and help researchers and physicians quickly identify relevant publications, we developed BioLitMine, an advanced literature mining tool that takes advantage of the medical subject heading (MeSH) index and gene-to-publication annotations already available for PubMed literature. Using BioLitMine, a user can identify what MeSH terms are represented in the set of publications associated with a given gene of the interest, or start with a term and identify relevant publications. Users can also use the tool to find co-cited genes and a build a literature co-citation network. In addition, BioLitMine can help users build a gene list relevant to a MeSH terms, such as a list of genes relevant to "stem cells" or "breast neoplasms." Users can also start with a gene or pathway of interest and identify authors associated with that gene or pathway, a feature that makes it easier to identify experts who might serve as collaborators or reviewers. Altogether, BioLitMine extends the value of PubMed-indexed literature and its existing expert curation by providing a robust and gene-centric approach to retrieval of relevant information.
Baolong Xia, Gabriel Amador, Raghuvir Viswanatha, Jonathan Zirin, Stephanie E Mohr, and Norbert Perrimon. 2020. “CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells.” Nat Protoc, 15, 10, Pp. 3478-3498.Abstract
Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes.
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.