Thinking ahead--What could custom CRISPR engineered cell lines do for your research?

Open call to the Drosophila research community from the DRSC:

Since it was founded in 2003 by Prof. N. Perrimon, the Drosophila RNAi Screening Center (DRSC) has served as a technology transfer center, helping the Drosophila community-at-large gain access to leading-edge technologies such as genome-wide RNAi.

As readers of this blog are likely aware, we have been working to support technology transfer in many areas additional to RNAi, including in the area of CRISPR-Cas9 engineering. For example, we developed and made freely available a database of short guide RNAs, accompanying genome browser-based online user interface, and sgRNA efficiency prediction tool to help support sgRNA selection for CRISPR-Cas9 engineering in flies (see www.flyrnai.org/crispr2).

The DRSC will apply early next year (end of February 2015) for renewal of our NIH R01 grant funding, which makes it possible for us to provide all we do to the community.

One of the things we are beginning to do with community members, and would like to propose to expand and continue in the next funding cycle, is to build custom CRISPR-Cas9-modified cell lines. These can be of value for a wide range of studies, including but not limited to RNAi screens using custom engineered cells (e.g. knockout mutant cells for sensitized screens, endogenously tagged loci for reporter assays or to screen for disruption of sub-cellular localization).

In short, we need your help!

If custom engineered cells would help your research--i.e. if you can imagine turning to the DRSC to help support making and/or screening of specific custom lines for your research within the next, say, 1-3 years, and particularly if you're a US-based lab, we would appreciate if you'd please get in touch and be willing to write a letter of support for our renewal application. We are of course also interested to hear from folks who are planning to use our library resources for other types of screens in the next few years, and from others who are depending on continuity of online resources and/or research services at the DRSC. Ideas for additional tools or resources are also welcome.

Please contact, or have your PI contact, DRSC Director (and blog author) Dr. Stephanie Mohr.

Review and detailed protocols--CRISPR-Cas9 in flies and fly cells

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.  

From the abstract: "... we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals."

Includes helpful tables listing sgRNA design tools, relevant fly stocks and plasmids.

Cross-species cell-based study of the lipoprotein lipase-binding protein GPIHBP1

Beigneux AP, Fong LG, Bensadoun A, Davies BS, Oberer M, Gårdsvoll H, Ploug M, Young SG. GPIHBP1 Missense Mutations Often Cause Multimerization of GPIHBP1 and Thereby Prevent Lipoprotein Lipase Binding. Circ Res. 2014 Nov 11. pii: CIRCRESAHA.114.305085. PMID: 25387803.

From the abstract: "GPIHBP1, a GPI-anchored protein of capillary endothelial cells, binds lipoprotein lipase (LPL) in the subendothelial spaces and shuttles it to the capillary lumen. GPIHBP1 missense mutations that interfere with LPL binding cause familial chylomicronemia. ... We expressed mutant forms of GPIHBP1 in Chinese hamster ovary cells, rat and human endothelial cells, and Drosophila S2 cells. In each expression system, mutation of cysteines in GPIHBP1's Ly6 domain (including mutants identified in chylomicronemia patients) led to the formation of disulfide-linked dimers and multimers. ..."

in vivo RNAi screen related to ALS

Deivasigamani S, Verma HK, Ueda R, Ratnaparkhi A, Ratnaparkhi GS. A genetic screen identifies Tor as an interactor of VAPB in a Drosophila model of amyotrophic lateral sclerosis. Biol Open. 2014 Oct 31. pii: BIO201410066. PMID: 25361581.

From the abstract: "Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder characterized by selective death of motor neurons. In 5-10% of the familial cases, the disease is inherited because of mutations. One such mutation, P56S, was identified in human VAPB that behaves in a dominant negative manner, sequestering wild type protein into cytoplasmic inclusions. We have conducted a reverse genetic screen to identify interactors of Drosophila VAPB. We screened 2635 genes and identified 103 interactors, of which 45 were enhancers and 58 were suppressors of VAPB function. Interestingly, the screen identified known ALS loci - TBPH, alsin2 and SOD1. Also identified were genes involved in cellular energetics and homeostasis which were used to build a gene regulatory network of VAPB modifiers. One key modifier identified was Tor, ..."

The biology of RNAi--recent studies

Bronkhorst AW, van Cleef KW, Venselaar H, van Rij RP. A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response. Nucleic Acids Res. 2014 Oct 29;42(19):12237-48. PMID: 25274730.

From the abstract: "... Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. ... Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response."

Gandhi SG, Bag I, Sengupta S, Pal-Bhadra M, Bhadra U. Drosophila oncogene Gas41 is RNAi modulator that intersects heterochromatin and siRNA pathway. FEBS J. 2014 Oct 16. PMID: 25323651.

From the abstract: "... These findings suggest that, Drosophila Gas41 guides the repeat associated gene silencing, and Dicer1 interaction thereby depicting a new role of the Gas41. ... In Drosophila, Gas41 plays a dual role. In one hand, it seems to participate with Dicer 1 in the RNAi pathway and alternatively also participate in repeat-induced gene silencing by accumulating heterochromatin proteins at the mw array promoters. ..."

Cell-based assays--new reports

Ramdas NM, Shivashankar GV. Cytoskeletal Control of Nuclear Morphology and Chromatin Organization. J Mol Biol. 2014 Oct 2. pii: S0022-2836(14)00495-1. PMID: 25281900.

From the abstract: "... We demonstrate here the differential influence of perinuclear actin- and microtubule-driven assemblies on nuclear architecture using pharmacological inhibitors and targeted RNA interference knockdown of cytoskeleton components in Drosophila cells. We find evidence that the loss of perinuclear actin assembly results in basolateral enhancement of microtubule organization and this is reflected functionally by enhanced nuclear dynamics. ..."

Ribeiro SA, D'Ambrosio MV, Vale RD. Induction of Focal Adhesions and Motility in Drosophila S2 cells. Mol Biol Cell. 2014 PMID: 25273555.

From the abstract: "... Here, we describe a system for inducing the formation of focal adhesions in normally non-ECM-adherent, non-motile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a non-polarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. ... These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility."

Genome-wide RNAi screen related to Parkinson's disease

Ivatt RM, Sanchez-Martinez A, Godena VK, Brown S, Ziviani E, Whitworth AJ. Genome-wide RNAi screen identifies the Parkinson disease GWAS risk locus SREBF1 as a regulator of mitophagy. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8494-9. PMID: 24912190; PMCID: PMC4060696.

From the abstract: "Genetic analysis of Parkinson disease (PD) has identified several genes whose mutation causes inherited parkinsonism, as well as risk loci for sporadic PD. PTEN-induced kinase 1 (PINK1) and parkin, linked to autosomal recessive PD, act in a common genetic pathway regulating the autophagic degradation of mitochondria, termed mitophagy. We undertook a genome-wide RNAi screen as an unbiased approach to identify genes regulating the PINK1/Parkin pathway. We identified several genes that have a conserved function in promoting mitochondrial translocation of Parkin and subsequent mitophagy, most notably sterol regulatory element binding transcription factor 1 (SREBF1), F-box and WD40 domain protein 7 (FBXW7), and other components of the lipogenesis pathway. ..."

in vivo genome-wide screen--NURF and dREAM

Bohla D, Herold M, Panzer I, Buxa MK, Ali T, Demmers J, Krüger M, Scharfe M, Jarek M, Bartkuhn M, Renkawitz R. A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking. PLoS One. 2014 Sep 23;9(9):e107765. PMID: 25247414; PMCID: PMC4172637.

From the abstract: "... We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident ..."

in vivo genome-wide RNAi screen--regulation of synapse function

Dani N, Zhu H, Broadie K. Two protein N-acetylgalactosaminyl transferases regulate synaptic plasticity by activity-dependent regulation of integrin signaling. J Neurosci. 2014 Sep 24;34(39):13047-65. PMID: 25253852; PMCID: PMC4172800.

From the abstract: "Using a Drosophila whole-genome transgenic RNAi screen for glycogenes regulating synapse function, we have identified two protein α-N-acetylgalactosaminyltransferases (pgant3 and pgant35A) that regulate synaptic O-linked glycosylation (GalNAcα1-O-S/T). Loss of either pgant alone elevates presynaptic/postsynaptic molecular assembly and evoked neurotransmission strength, but synapses appear restored to normal in double mutants. ..."

Defining the cell shape space

Sailem H, Bousgouni V, Cooper S, Bakal C. Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity. Open Biol. 2014 Jan 22;4:130132. PMID: 24451547; PubMed Central PMCID: PMC3909273.

  From the abstract: "... We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. ... We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations."

Perspectives paper in Genetics--"Publication Trends in Model Organism Research"

This perspectives paper in Genetics, "Publication Trends in Model Organism Research," seems of general interest to those of us in the Drosophila functional genomics research field and beyond.

Full-genome screen related to functional insulators

Another full-genome DRSC screen published--congrats!

Fig. 1 from the Bohla et al. paper.

Bohla D, Herold M, Panzer I, Buxa MK, Ali T, Demmers J, Krüger M, Scharfe M, Jarek M, Bartkuhn M, Renkawitz R. A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking. PLoS One. 2014 Sep 23;9(9):e107765. PubMed PMID: 25247414; PubMed Central PMCID: PMC4172637.

From the abstract: "... Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. ..."

G3 report--two-step CRISPR approach

Another fly gene editing paper early online at G3--

Xu Zhang, Wouter H. Koolhaas and Frank Schnorrer (2014) A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient Genome Engineering in Drosophila. G3. Early online.

G3 report--plasmids for CRISPR-Cas9 in flies

Gokcezade J, Sienski G, Duchek P. Efficient CRISPR/Cas9 Plasmids for Rapid and Versatile Genome Editing in Drosophila. G3 (Bethesda). 2014 Sep 17. pii: g3.114.014126. PMID: 25236734.

In the Acknowledgements the authors note that "The vectors described in this study (pDCC5 and pDCC6) will be available from Addgene and the Vienna Drosophila Resource Center (VDRC).

Localization of CPTI collection of YFP-trap proteins

Lye CM, Naylor HW, Sanson B. Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos. Development. 2014 Oct;141(20):4006-17. PMID: 25294944.

From the abstract:  "... The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. ..."

MiMIC-based approach to gene knock-in

Early online at G3 --

Sven Vilain, Roeland Vanhauwaert, Ine Maes, Nils Schoovaerts, Lujia Zhou, Sandra Soukup, Raquel da Cunha, Elsa Lauwers, Mark Fiers, and Patrik Verstreken Fast and Efficient Drosophila melanogaster Gene Knock-Ins Using MiMIC Transposons
G3 early online October 8, 2014,
http://www.g3journal.org/content/early/2014/10/08/g3.114.014803.abstract

Drosophila cell-based genome-wide screen related to Parkinson's disease

Ivatt RM, Sanchez-Martinez A, Godena VK, Brown S, Ziviani E, Whitworth AJ. Genome-wide RNAi screen identifies the Parkinson disease GWAS risk locus SREBF1 as a regulator of mitophagy. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8494-9. PMID: 24912190; PubMed Central PMCID: PMC4060696.  

From the abstract: "... We undertook a genome-wide RNAi screen as an unbiased approach to identify genes regulating the PINK1/Parkin pathway. We identified several genes that have a conserved function in promoting mitochondrial translocation of Parkin and subsequent mitophagy, most notably sterol regulatory element binding transcription factor 1 (SREBF1), F-box and WD40 domain protein 7 (FBXW7), and other components of the lipogenesis pathway. ..."

Drosophila Models of Human Disease: Large-scale study of genes on the fly X-chromosome...

Drosophila Models of Human Disease: Large-scale study of genes on the fly X-chromosome...: Yamamoto S, Jaiswal M, Charng WL, Gambin T, Karaca E, Mirzaa G, Wiszniewski W, Sandoval H, Haelterman NA, Xiong B, Zhang K, Bayat V, David G...

Study related to Abl kinase uses primary cultured cells

This is not an RNAi study but the approach taken might of interest to those thinking about cell-based studies. Liu L, Wu CF. Distinct effects of Abelson kinase mutations on myocytes and neurons in dissociated Drosophila embryonic cultures: mimicking of high temperature. PLoS One. 2014 Jan 21;9(1):e86438. PMID: 24466097; PMCID: PMC3897706. From the abstract: "... we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos ..."

Protocol paper -- RNAi and other techniques in gut-microbe studies

Houtz PL, Buchon N. Methods to Assess Intestinal Stem Cell Activity in Response to Microbes in Drosophila melanogaster. Methods Mol Biol. 2014;1213:171-82. PMID: 25173382.

Protocol paper includes information about RNAi in the germline

DeVorkin L, Gorski SM. Genetic manipulation of autophagy in the Drosophila ovary. Cold Spring Harb Protoc. 2014 Sep 2;2014(9):pdb.prot080358. PMID: 25183818. From the abstract: "... Here we describe how to generate a homozygous mutant germline using the FLP-DFS (dominant female sterile) technique, how to generate somatic clones, and how to induce targeted gene knockdown in the germline using RNAi."

UAS system combined with CRISPR-Cas9 in Drosophila

Zhaoyu Xue, Menghua Wu, Kejia Wen, Menda Ren, Li Long, Xuedi Zhang and Guanjun Gao. CRISPR/Cas9 Mediates Efficient Conditional Mutagenesis in Drosophila. G3. Available as early online content.

From the abstract:  "... tissue-specific expression of Cas9 driven by the Gal4/upstream activating site (UAS) system with various ubiquitously expressed gRNA transgenes to effectively inactivate gene expression in a temporally and spatially controlled manner. ... by including multiple gRNAs in a transgenic vector to target a single gene, we achieved a high degree of gene mutagenesis in specific tissues. ..."

Genome editing--design tools

On my desk to read today--another paper related to CRISPRs.

Montague TG, Cruz JM, Gagnon JA, Church GM, Valen E. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 2014 Jul;42(Web Server issue):W401-7. PMID: 24861617.

This report describes the ChopChop suite of tools, which includes support for Drosophila sgRNA design.

The DRSC offers our CRISPR2 sgRNA design tool, which provides a genome-browse view and includes efficiency scores (or you can run any designs through a 'calculator').

Additional tools are listed at AddGene.

The DRSC offers support for sgRNA and donor construct building through the Genome Engineering Production Group.

RNAi screening in fly cells to investigate Brucella infection

Pandey A, Ding SL, Ficht TA, de Figueiredo P. siRNA Screens Using Drosophila Cells to Identify Host Factors Required for Infection. Methods Mol Biol. 2014;1197:229-44. PMID: 25172284.

Fly CRISPR -- fluorescent tag and RMCE approach

Learned about this pre-print online from the Insect Genetic Technologies Research Coordination Network (see "technology tips" section).

Describes use of fluorescent tagging to more efficiently identify successful knock-ins, then RMCE to change the knocked-in cassette.

Xu Zhang, Wouter Koolhaas, Frank Schnorrer. A versatile two-step CRISPR- and RMCE-based strategy for efficient genome engineering in Drosophila. doi: http://dx.doi.org/10.1101/007864

Pre-print is available online:
http://biorxiv.org/content/early/2014/08/11/007864

Anti-Cas9 antibody

If an anti-Cas9 antibody would be useful to your CRISPR-related projects--at least one company now reports availability of an anti-Cas9 monoclonal.

Integer linear optimization approach to signaling network construction based on protein-protein interaction and RNAi data sets

Ozsoy OE, Can T. A divide and conquer approach for construction of large-scale signaling networks from PPI and RNAi data using linear programming. IEEE/ACM Trans Comput Biol Bioinform. 2013 Jul-Aug;10(4):869-83. PMID: 24334382.

From the abstract: "... In this paper, we propose an integer linear optimization (ILP) model for reconstruction of signaling networks from RNAi data and a reference network. ..."

Related approaches available as online tools at the DRSC include COMPLEAT and SignedPPI

New report--CRISPR-Cas9 nickase system in Drosophila

Ren X, Yang Z, Mao D, Chang Z, Qiao HH, Wang X, Sun J, Hu Q, Cui Y, Liu LP, Ji JY, Xu J, Ni JQ. Performance of the Cas9 Nickase System in Drosophila melanogaster. G3 (Bethesda). 2014 Aug 15. pii: g3.114.013821. PMID: 25128437.

in vivo fly RNAi screen uses live imaging assay

Mauri F, Reichardt I, Mummery-Widmer JL, Yamazaki M, Knoblich JA. The Conserved Discs-large Binding Partner Banderuola Regulates Asymmetric Cell Division in Drosophila. Curr Biol. 2014 Jul 30. pii: S0960-9822(14)00775-1. PMID: 25088559.

Study explores culture media for Drosophila cells

Burnette M, Brito-Robinson T, Li J, Zartman J. An inverse small molecule screen to design a chemically defined medium supporting long-term growth of Drosophila cell lines. Mol Biosyst. 2014 Aug 6. PMID: 25096480.

From the abstract: "Drosophila cell culture is used as a model system with multiple applications ...  To characterize the minimal requirements for long-term maintenance of Drosophila cell lines, we developed an inverse screening strategy to identify small molecules and synergies stimulating proliferation in a chemically defined medium. ... Validated factors were investigated for their ability to maintain cell growth over multiple passages in the chemically defined medium (CDM). The polyamine spermidine proved to be the critical component that enables the CDM to support long-term maintenance of Cl.8 cells. Spermidine supplementation upregulates DNA synthesis for Cl.8 and S2 cells and increases MAPK signaling for Cl.8 cells. The CDM also supports the long-term growth of Kc167 cells. Our target scoring approach validated the importance of polyamines ... Future iterations of the screen will enable the identification of compound combinations optimized for specific applications ... thus increasing the versatility of Drosophila cell culture as both a genetic and biochemical model system. ..."

Planning Gal4-UAS studies in the larval CNS, such as with in vivo RNAi? Report of >6,000 Gal4 lines

Li HH, Kroll JR, Lennox SM, Ogundeyi O, Jeter J, Depasquale G, Truman JW. A GAL4 Driver Resource for Developmental and Behavioral Studies on the Larval CNS of Drosophila. Cell Rep. 2014 Jul 30. pii: S2211-1247(14)00569-5. PMID: 25088417.

Genome-wide, high-content image-based screen preformed at DRSC--identification of regulators of organization of polycomb foci

Gonzalez I, Mateos-Langerak J, Thomas A, Cheutin T, Cavalli G. Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen. Mol Cell. 2014 May 8;54(3):485-99. PMID: 24703951.  

From the abstract: "... Here, we report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. ... suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci."

Cell-based assays related to Notch signaling

Xu A, Irvine KD. Notch-ligand binding assays in Drosophila cells. Methods Mol Biol. 2014;1187:277-84. PMID: 25053497.

 Li J, Housden BE, Bray SJ. Notch signaling assays in Drosophila cultured cell lines. Methods Mol Biol. 2014;1187:131-41. PMID: 25053486.

RNA biology--viruses & RNAi suppression

van Mierlo JT, Overheul GJ, Obadia B, van Cleef KW, Webster CL, Saleh MC, Obbard DJ, van Rij RP. Novel Drosophila Viruses Encode Host-Specific Suppressors of RNAi. PLoS Pathog. 2014 Jul 7;10(7):e1004256. PMID: 25032815.

Genetic manipulation of both host cells and pathogen--"iMAD" protocol

O'Connor TJ, Isberg RR. iMAD, a genetic screening strategy for dissecting complex interactions between a pathogen and its host. Nat Protoc. 2014 Aug;9(8):1916-30. PMID: 25033208.

From the abstract: "... Here we present a protocol for dissecting the interaction between a pathogen (Legionella pneumophila) and its host (cultured Drosophila melanogaster cells) using bacterial mutagenesis and host RNAi. ..."

Genome-wide RNAi screen identifies neurons involved in female receptivity behavior

Bussell JJ, Yapici N, Zhang SX, Dickson BJ, Vosshall LB. Abdominal-B Neurons Control Drosophila Virgin Female Receptivity. Curr Biol. 2014 Jul 3. pii: S0960-9822(14)00689-7. PMID: 24998527.

From the abstract: "... Using a genome-wide neuronal RNAi screen, we identify a subpopulation of neurons responsible for pausing, a novel behavioral aspect of virgin female receptivity characterized in this study. ..."

in vivo RNAi screen related to Notch signaling in follicle cells

Xie G, Yu Z, Jia D, Jiao R, Deng WM. E(y)1/TAF9 mediates the transcriptional output of Notch signaling in Drosophila. J Cell Sci. 2014 Jul 11. PMID: 25015288.

From the abstract: "... We identified E(y)1/TAF9 in a large-scale in vivo RNAi screen for genes involved in a Notch-dependent mitotic-to-endocycle transition in Drosophila follicle cells. Knockdown of e(y)1/TAF9 displayed Notch-like phenotypes ..."

Fly RNAi screen of about 150 genes included in obesity study

Lee JH, Bassel-Duby R, Olson EN. Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 1;111(26):9491-6. PMID: 24979807.

Genome-wide RNAi screen in fly and mammalian systems related to Parkinson's disease

Ivatt RM, Whitworth AJ. SREBF1 links lipogenesis to mitophagy and sporadic Parkinson disease. Autophagy. 2014 Jun 27;10(8). Review. PubMed PMID: 24991824.

From the abstract: "Mitochondrial quality control has an impact on many diseases, but intense research has focused on the action of 2 genes linked to heritable forms of Parkinson disease (PD), PINK1 and PARK2/parkin, which act in a common pathway to promote mitophagy. However, criticism has been raised that little evidence links this mechanism to sporadic PD. To gain a greater insight into the mechanisms of PINK1-PARK2 mediated mitophagy, we undertook a genome-wide RNAi screen in Drosophila and human cell models. ... Our findings suggest a role for lipid synthesis in PINK1-PARK2 mediated mitophagy, and propose a mechanistic link between familial and sporadic PD, supporting a common etiology."

DRSC and collaborators present Online GESS

Yilmazel B, Hu Y, Sigoillot F, Smith JA, Shamu CE, Perrimon N, Mohr SE. Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis. BMC Bioinformatics. 2014 Jun 17;15:192. PMID: 24934636; PMCID: PMC4073188.

From the abstract: "... Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences."

Wing blisters as model for tissue morphogenesis--in vivo RNAi study

Bilousov O, Koval A, Keshelava A, Katanaev VL. Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases. PLoS One. 2014 Jun 26;9(6):e101133. PMID: 24968325; PMCID: PMC4072764. From the abstract: "... Here we focused on Drosophila wing tissue morphogenesis, a fairly complex developmental program ... By means of RNAi-silencing technique and the blister phenotype as readout, we identify numerous novel proteins potentially involved in wing sheet adhesion ..."

in vivo fly RNAi screen looks at cytoskeletal and other components in muscle

Perkins AD, Tanentzapf G. An Ongoing Role for Structural Sarcomeric Components in Maintaining Drosophila melanogaster Muscle Function and Structure. PLoS One. 2014 Jun 10;9(6):e99362. PMID: 24915196; PMCID: PMC4051695.

in vivo fly RNAi kinase and phosphatase screen looks at synapse

Bulat V, Rast M, Pielage J. Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2. J Cell Biol. 2014 Jan 6;204(1):77-94. PMID: 24395637; PMCID: PMC3882785.

in vivo RNAi screen related to cell death points to CDK7

Morishita J, Kang MJ, Fidelin K, Ryoo HD. CDK7 regulates the mitochondrial localization of a tail-anchored proapoptotic protein, Hid. Cell Rep. 2013 Dec 26;5(6):1481-8. PMID: 24360962; PMCID: PMC3892150.

Running oranelles on an S2 cell track (with or without RNAi treatment)

Lu W, Del Castillo U, Gelfand VI. Organelle transport in cultured Drosophila cells: S2 cell line and primary neurons. J Vis Exp. 2013 Nov 20;(81):e50838. PMID: 24300413.

Biology of the RNAi pathway--Argonaute-mediated microRNA biogenesis

Yang JS, Smibert P, Westholm JO, Jee D, Maurin T, Lai EC. Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila. Nucleic Acids Res. 2014 Feb;42(3):1987-2002. PMID: 24220090; PMCID: PMC3919586.  

From the abstract: "Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. ... Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates."

Follow-up on RNAi screen links SCF(Skp2) and Double-parked

Kroeger PT Jr, Shoue DA, Mezzacappa FM, Gerlach GF, Wingert RA, Schulz RA. Knockdown of SCF(Skp2) function causes double-parked accumulation in the nucleus and DNA re-replication in Drosophila plasmatocytes. PLoS One. 2013 Oct 24;8(10):e79019. PMID: 24205363; PMCID: PMC3812016.

RNAi targeting of 270 fly orthologs of human genes helps reveal conserved functional networks related to intellectual disability

See this post in Drosophila Models of Human Disease.

New fly CRISPR report

Fillip Port (a past DRSC screener) and colleagues have published a new report on CRISPRs in flies. Additional data included as compared with their online preprint.

Port F, Chen HM, Lee T, Bullock SL. Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila. Proc Natl Acad Sci U S A. 2014 Jul 7. pii: 201405500. PMID: 25002478.

GenomeRNAi at DKFZ announces version 13

Our colleagues at the DKFZ recently announced that they have launched version 13 of GenomeRNAi. Their news item on the release indicates that the database "now contains phenotype data from 194 human RNAi screens and 183 screens in D. melanogaster" and that the "number of gene-phenotype associations has increased to over 950,000." Check it out!

Protocol report on transgenic ORF strains

Bischof J, Sheils EM, Björklund M, Basler K. Generation of a transgenic ORFeome library in Drosophila. Nat Protoc. 2014 Jul;9(7):1607-20. PMID: 24922270.

From the abstract: "Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by upstream activation sequences (UAS sites) ... We also provide details for FLP/FRT-mediated in vivo exchange of epitope tags (or regulatory regions) in the ORF library strains, which further extends the potential applications of the library. These transgenic UAS-ORF strains are a useful resource to complement and validate genetic experiments performed with loss-of-function mutants and RNA interference (RNAi) lines."

Large-scale in vivo Drososophila RNAi used in study of germ cells

Jankovics F, Henn L, Bujna A, Vilmos P, Spirohn K, Boutros M, Erdélyi M. Functional analysis of the Drosophila embryonic germ cell transcriptome by RNA interference. PLoS One. 2014 Jun 4;9(6):e98579. PMID: 24896584.

Large-scale in vivo RNAi screen in glia

Meyer S, Schmidt I, Klämbt C. Glia ECM interactions are required to shape the Drosophila nervous system. Mech Dev. 2014 May 21. pii: S0925-4773(14)00027-6. PMID: 24859129.

Report of using purified Cas9 for CRISPR genome engineering in Drosophila

Lee JS, Kwak SJ, Kim J, Noh HM, Kim JS, Yu K. RNA-Guided Genome Editing in Drosophila with the Purified Cas9 Protein. G3 (Bethesda). 2014 May 28. pii: g3.114.012179. PMID: 24875628.

Abstract: "We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay."

Review--Functional Genomics Resources

My co-authors and I are pleased to present this Genetics journal "toolbox" series review article.

Mohr SE, Hu Y, Kim K, Housden BE, Perrimon N. Resources for Functional Genomics Studies in Drosophila melanogaster. Genetics. PubMed PMID: 24653003.

Please note that the final published version is now available--complete with live links to the many websites and online software tools we refer to.

Genome-wide in vivo RNAi screen for genes involved in epithelial development

Berns N, Woichansky I, Friedrichsen S, Kraft N, Riechmann V. A genome-scale in vivo RNAi analysis of epithelial development in Drosophila identifies new proliferation domains outside of the stem cell niche. J Cell Sci. 2014 Apr 24. PMID: 24762813.

New report on using CRISPR/Cas9 in Drosophila cells

Böttcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Förstemann K. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res. 2014 Apr 19. PMID: 24748663.

From the abstract:  "We present a series of PCR template vectors for C-terminal protein tagging and clonal Drosophila S2 cell lines with stable expression of a myc-tagged cas9 protein." The authors also state that the plasmid constructs associated with the publication will be available at AddGene.

Recent review of in vivo RNAi screening

Yamamoto-Hino M, Goto S. In Vivo RNAi-Based Screens: Studies in Model Organisms. Genes (Basel). 2013 Nov 25;4(4):646-65. PMID: 24705267.

in vivo screen of about 300 genes related to the 'periodic transcriptome'

Liang L, Haug JS, Seidel CW, Gibson MC. Functional Genomic Analysis of the Periodic Transcriptome in the Developing Drosophila Wing. Dev Cell. 2014 Mar 27. pii: S1534-5807(14)00134-8. PMID: 24684830.

Spindle assembly screen of 96 candidate genes

Gallaud E, Caous R, Pascal A, Bazile F, Gagné JP, Huet S, Poirier GG, Chrétien D, Richard-Parpaillon L, Giet R. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells. J Cell Biol. 2014 Mar 31;204(7):1111-21. PMID: 24687279.

From the abstract: "... To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. ..."

Review--RNAi screening and signaling networks

Evans L, Sailem H, Vargas PP, Bakal C. Inferring signalling networks from images. J Microsc. 2013 Oct;252(1):1-7. PMID: 23841886.

From the abstract:  "This review describes the methodology used to map signalling networks using data generated in the context of RNAi screens."

Detailed methods report for TALEN-based genome engineering in flies

Zhang X, Ferreira IR, Schnorrer F. A simple TALEN-based protocol for efficient genome-editing in Drosophila. Methods. 2014 Mar 26. pii: S1046-2023(14)00122-4. PMID: 24680697.

Arthropod Genomics 2014

Passing along an announcement for Arthropod Genomics 2014. The deadline to submit abstracts is April 17, 2014, and the deadline for early registration is April 21, 2014.

Luciferase-based assays in cultured cells

Yun C, Dasgupta R. Luciferase reporter assay in Drosophila and Mammalian tissue culture cells. Curr Protoc Chem Biol. 2014 Mar 14;6(1):7-23. PMID: 24652620.

Not RNAi -- Methods chapter on other ways to interrogate gene function in flies

Venken KJ, Bellen HJ. Chemical mutagens, transposons, and transgenes to interrogate gene function in Drosophila melanogaster. Methods. 2014 Feb 28. pii: S1046-2023(14)00069-3. PMID: 24583113.

Single-cell analysis of S2R+ RNAi screen image data

Dey G, Gupta GD, Ramalingam B, Sathe M, Mayor S, Thattai M. Exploiting cell-to-cell variability to detect cellular perturbations. PLoS One. 2014 Mar 4;9(3):e90540. PMID: 24594940; PMCID: PMC3942435.

DRSC & TRiP at ADRC

Are you at the ADRC? So are we. Posters 855C and 857B.

New CRISPR tools at DRSC--Find CRISPR2 and Efficiency Tool

Interested in CRISPRs in flies? Check out the new version of our Find CRISPRs tool, Find CRISPRs Version 2, and new Efficiency Predictor that assigns a score to a given short guide RNA sequence.

The DRSC offers CRISPR design help, CRISPR sgRNA construct production and donor construct production. You can view services and our low prices, as well as place requests, at the website of the new Genome Engineering Production Group at the DRSC at HMS. We can send custom-made constructs to you or directly to a commercial Drosophila injection company.

CRISPR in flies with novel non-coding gene promoter driving expression of the short guide RNA

New report in G3: Xue, Ren, Wu, Dai, Rong and Gao. Efficient Gene Knock-Out and Knock-In with Transgenic Cas9 in Drosophila. Early online access.

Use of fly cells to study the cytoskeleton

Nye J, Buster DW, Rogers GC. The use of cultured Drosophila cells for studying the microtubule cytoskeleton. Methods Mol Biol. 2014;1136:81-101. PMID: 24633795.

From the abstract:  The authors "describe basic techniques for gene knockdown, transgene expression, preparation for fluorescence microscopy, and centrosome enrichment using cultured Drosophila cells with an emphasis on studying the microtubule cytoskeleton."

Full-genome RNAi screen in S2 cells looks at RAS/MAPK signaling

Ashton-Beaucage D, Udell CM, Gendron P, Sahmi M, Lefrançois M, Baril C, Guenier AS, Duchaine J, Lamarre D, Lemieux S, Therrien M. A Functional Screen Reveals an Extensive Layer of Transcriptional and Splicing Control Underlying RAS/MAPK Signaling in Drosophila. PLoS Biol. 2014 Mar 18;12(3):e1001809. PMID: 24643257.

Use of tissue-specific RNAi to help assess "biological quality" in RNA-seq data

Amaral AJ, Brito FF, Chobanyan T, Yoshikawa S, Yokokura T, Van Vactor D, Gama-Carvalho M. Quality assessment and control of tissue specific RNA-seq libraries of Drosophila transgenic RNAi models. Front Genet. 2014 Mar 5;5:43. PMID: 24634674.

Life in a bubble

At the DRSC we recently tested a new commercial plate type supporting growth of cells in very small volumes of media--tiny domes of liquid in 96- or 384-well format. We have not had time for much testing but we can say this:  S2R+ cultured Drosophila cells grew just fine in this format in our hands. Photo below. Feel free to email the DRSC Director if you have any questions. DropArray plates from Curiox. Posting this for information purposes only (should not be considered an endorsement).

Large-scale in vivo RNAi screen for factors related to germline stem cells

Yan D, Neumüller RA, Buckner M, Ayers K, Li H, Hu Y, Yang-Zhou D, Pan L, Wang X, Kelley C, Vinayagam A, Binari R, Randklev S, Perkins LA, Xie T, Cooley L, Perrimon N. A regulatory network of Drosophila germline stem cell self-renewal. Dev Cell. 2014 Feb 24;28(4):459-73. PMID: 24576427.

From the abstract: "... To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. ..."

These authors deposited screen data into RSVP, the TRiP's RNAi Stock Validation and Phenotypes data repository. Additional data submissions welcome!  Check it out online here.

Methods paper on fly RNAi in cells and in vivo

Humbly offered by your blog author ...


Mohr SE. RNAi screening in Drosophila cells and in vivo. Methods. 2014 Feb 24. pii: S1046-2023(14)00062-0. PMID: 24576618.

Interplay of RNAi, cell death and tranposons

Birchler JA. Does ectopic cell death cause somatic mutations in the neighboring cells by activating transposons? Mob Genet Elements. 2014 Jan 1;4(1):e28040. PMID: 24567848.

Genome-wide RNAi screen for host factors related to West Nile Virus

Yasunaga A, Hanna SL, Li J, Cho H, Rose PP, Spiridigliozzi A, Gold B, Diamond MS, Cherry S. Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection. PLoS Pathog. 2014 Feb 13;10(2):e1003914. PMID: 24550726; PMCID: PMC3923753.

From the abstract:  "Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts."

A correspondence in Nature Methods raises concerns regarding in vivo RNAi fly stocks

Green et al. (2014) A Drosophila RNAi collection is subject to dominant phenotypic effects. Nature Methods 11, 222–223.

in vivo screen looks at role of RNA binding proteins in neuron morphogenesis

Olesnicky EC, Killian DJ, Garcia E, Morton MC, Rathjen AR, Sola IE, Gavis ER. Extensive Use of RNA Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis. G3 (Bethesda). 2013 Dec 20. pii: g3.113.009795v2. PMID: 24347626.

From the abstract:  "... we conducted an RNAi screen to identify Drosophila proteins annotated as either RNA binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 such proteins whose knock-down resulted in aberrant dendritic morphology ..."

in vivo RNAi screen related to fat storage

Baumbach J, Hummel P, Bickmeyer I, Kowalczyk KM, Frank M, Knorr K, Hildebrandt A, Riedel D, Jäckle H, Kühnlein RP. A Drosophila in vivo screen identifies store-operated calcium entry as a key regulator of adiposity. Cell Metab. 2014 PMID: 24506874.

New cell image analysis tool reported

Tsygankov D, Bilancia CG, Vitriol EA, Hahn KM, Peifer M, Elston TC. CellGeo: A computational platform for the analysis of shape changes in cells with complex geometries. J Cell Biol. 2014 Feb 3;204(3):443-60. PMID: 24493591.

DKFZ's GenomeRNAi releases v.12

The DKFZ's GenomeRNAi team recently announced release of a version 12 with new data sets and features. Check it out!

CRISPR knock-in approaches in Drosophila.

Scott J. Gratz, Fiona P. Ukken, C. Dustin Rubinstein, Gene Thiede, Laura K. Donohue, Alexander M. Cummings, and Kate M. O'Connor-Giles. Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila. Genetics genetics.113.160713; early online January 29, 2014

Methods review on genome engineering in flies--Updated 2-3-2014

Beumer KJ and Carroll D. Targeted genome engineering techniques in Drosophila. Methods. 2014 Jan 8. pii: S1046-2023(13)00455-6. PMID: 24412316.

2-3-2014 update: Another review on fly genome engineering approaches.

Bassett AR, Liu JL. CRISPR/Cas9 and Genome Editing in Drosophila. J Genet Genomics. 2014 Jan 20;41(1):7-19. PMID: 24480743.

To view all posts tagged "CRISPRs" click here.

Large-scale in vivo RNAi screen of conserved genes looks at glia

Ghosh A, Kling T, Snaidero N, Sampaio JL, Shevchenko A, Gras H, Geurten B, Göpfert MC, Schulz JB, Voigt A, Simons M. A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons. PLoS Genet. 2013 Dec;9(12):e1003980. doi: 10.1371/journal.pgen.1003980. PMID: 24348263; PMCID: PMC3861124.

Methods paper discusses in vivo RNAi for studying oogenesis

This new Methods journal paper from Hudson & Cooley on Drosophila oogenesis includes discussion of in vivo RNAi. Table 1 for example lists relevant Gal4 drivers useful for (among other things) RNAi-based interrogation of germline or somatic tissues in the fly ovary.

Hudson AM, Cooley L. Methods for studying oogenesis. Methods. 2014 Jan 16. pii: S1046-2023(14)00006-1. PMID: 24440745.

Methods paper on fly cell lines

This new paper in Methods from L. Cherbas & L. Gong should be a great resource for those interested in using Drosophila cells in research studies.

Cherbas L, Gong L. Cell lines. Methods. 2014 Jan 13. PMID: 24434506.

Shaken, then swirled.

On my desk to read today? This new report of a Drosophila cell-based RNAi screen for which the researchers used a mechanical assay--shake, swirl, shake--to interrogate cell-cell adhesion.

Toret CP, D'Ambrosio MV, Vale RD, Simon MA, Nelson WJ. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell-cell adhesion. J Cell Biol. 2014 Jan 20;204(2):265-79. PMID: 24446484.

GFP assay & innate immunity

This paper describes a new assay in S2 cells related to innate immunity.

Yang B, Lu A, Peng Q, Ling QZ, Ling E. Activity of fusion prophenoloxidase-GFP and its potential applications for innate immunity study. PLoS One. 2013 May 23;8(5):e64106. PMID: 23717543; PMCID: PMC3662757.