CRISPR/Cas9 in Drosophila cultured cells

RNAi screening in customized Drosophila cell lines has seemed like an inevitability since the emergence of the new genome engineering approaches.  With this report, that eventuality seems another step closer.

Bassett AR, Tibbit C, Ponting CP, Liu JL. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. Biol Open. 2013 Dec 10. PMID: 24326186.

Genome-wide S2 cell screen looking at centrosome-independent mitotic spindle assembly

Moutinho-Pereira S, Stuurman N, Afonso O, Hornsveld M, Aguiar P, Goshima G, Vale RD, Maiato H. Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells. Proc Natl Acad Sci U S A. 2013 Nov 19. PMID: 24255106.

From the abstract: "Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. ... Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells."

Argonaut-mediated miRNA biogenesis. Recent report.

Yang JS, Smibert P, Westholm JO, Jee D, Maurin T, Lai EC. Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila. Nucleic Acids Res. 2013 Nov 12. PMID: 24220090.

Drosophila CRISPR approaches--Cas9 in the germline

These two papers, one of which is co-authored by folks here at the DRSC, describe using germline expression of Cas9 for CRISPR genome engineering approaches.

Sebo ZL, Lee HB, Peng Y, Guo Y. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly (Austin). 2013 Oct 18;8(1). PMID: 24141137.

Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu LP, Yang Z, Mao D, Sun L, Wu Q, Ji JY, Xi J, Mohr SE, Xu J, Perrimon N, Ni JQ. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A. 2013 Nov 4. PMID: 24191015. Note: This paper includes a description of our searchable online resource of pre-computed CRISPR sgRNA designs.

Book in Methods in Molecular Biology series includes fly PIWI study methods

A current book in the Methods in Molecular Biology Series, PIWI-Interacting RNAs, includes protocols for Drosophila RNA-related research.

New fly TALEN paper.

Kondo T, Sakuma T, Wada H, Akimoto-Kato A, Yamamoto T, Hayashi S. TALEN-induced gene knock out in Drosophila. Dev Growth Differ. 2013 Oct 31. PMID: 24172335.

Interested to try TALENs?  The DRSC is offering custom TALEN constructs.

More new reports on fly genome engineering.

Sebo ZL, Lee HB, Peng Y, Guo Y. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering. Fly (Austin). 2013 Oct 18;8(1). PMID: 24141137.

Gratz SJ, Wildonger J, Harrison MM, O'Connor-Giles KM. CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly (Austin). 2013 Oct 2;7(4). PMID: 24088745.

Baena-Lopez LA, Alexandre C, Mitchell A, Pasakarnis L, Vincent JP. Accelerated homologous recombination and subsequent genome modification in Drosophila. Development. 2013 Oct 23. PMID: 24154526.

Click here to view all posts tagged CRISPRs.

DRSC DIOPT and DIOPT-DIST ortholog tools now include Xenopus and S. pombe

The DRSC is pleased to announce that our DIOPT and DIOPT-DIST tools for ortholog search now include Xenopus and S. pombe in addition to fly, worm, S. cerevisiae, zebrafish, mouse and human. We added these two new species in response to community feedback.

What's the difference between DIOPT and DIOPT-DIST?

DIOPT lets you search for orthologs for any pair of species (e.g. zebrafish orthologs of fly genes, or yeast orthologs of a mouse gene). Results from 10 different public tools/alorithms are shown.

DIOPT-DIST lets you search using any of the non-human species we report, and view not just putative human orthologs but also connections between those human orthologs and disease. Alternatively, you can start with a disease, view human genes associated with that disease and orthologs of those human genes in a given species of interest.

If you use these tools, please cite our paper:  Hu et al. BMC Bioinformatics. PMID: 21880147

DRSC online database of pre-computer CRISPR sgRNAs

The DRSC is pleased to announce the launch of a searchable database of pre-computed CRISPR sgRNA sequences targeting the fly genome. As always, your feedback is welcome.

GenomeRNAi launches 11.1 and asks for feedback

The folks at the DKFZ's GenomeRNAi have launched a version 11.1 and ask for feedback, in particular on the beta version of their 'screen comparison' tool.

in vivo screen of GPCRs that regulate flight. Recent report.

Agrawal T, Sadaf S, Hasan G. A Genetic RNAi Screen for IP(3)/Ca(2+) Coupled GPCRs in Drosophila Identifies the PdfR as a Regulator of Insect Flight. PLoS Genet. 2013 Oct;9(10):e1003849. PMID: 24098151; PMCID: PMC3789835.

 From the abstract: "... RNAi screen to identify GPCRs that regulate flight by activating the IP3 receptor ..."

Validation with RNAi in study of the Hippo pathway interactome. Recent report.

Kwon Y, Vinayagam A, Sun X, Dephoure N, Gygi SP, Hong P, Perrimon N. The Hippo Signaling Pathway Interactome. Science. 2013 Oct 10. PMID: 24114784.

From the abstract: "... a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNAi regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively."

Validation with RNAi in study of sensory neurons. Recent report.

Iyer EP, Iyer SC, Sullivan L, Wang D, Meduri R, Graybeal LL, Cox DN. Functional genomic analyses of two morphologically distinct classes of Drosophila sensory neurons: post-mitotic roles of transcription factors in dendritic patterning. PLoS One. 2013 PMID: 23977298; PMCID: PMC3744488.

From the abstract: "We have used a combination of cell-type specific transcriptional expression profiling coupled to a targeted and systematic in vivo RNAi functional validation screen."

Fly RNAi & Huntingtons disease.

The paper displayed at this post at Drosophila Models of Human Diseases fits here as well--Lu and colleagues describe the results of genome-wide RNAi screens in fly cells plus in vivo fly follow-up in a disease model mutant.

Another fly CRISPR report.

Early online at Genetics--this report from Kondo and Ueda on using CRISPRs in flies.

Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila. Genetics. Early online September 3, 2013, doi:10.1534/genetics.113.156737.

RNAi validation with a synthetic transcript

On my desk to read this week?

Jonchere V, Bennett D. Validating RNAi Phenotypes in Drosophila Using a Synthetic RNAi-Resistant Transgene. PLoS One. 2013;8(8):e70489. PMID: 23950943; PMCID: PMC3738578.

DRSC announces launch of FlyPrimerBank online resource

The DRSC is pleased to announce the launch of FlyPrimerBank.

FlyPrimerBank is built around a database of three precomputed qPCR primer pairs per Drosophila gene. Online search output includes information about the primer pairs and when available, validation test results from our group or the community at large.

For details please see our paper, just out in the journal G3. You can also check out the documentation page.

Important for RNAi studies, FlyPrimerBank can report overlap between the primers and RNAi reagents in public collections. This helps you avoid choosing primers that could amplify the reagent.

As always, your feedback on the online resource is welcome.

FlyPrimerBank also makes it possible for you to indicate which qPCR primers in FlyPrimerBank have or have not worked for them, or to upload the sequences of good primer pairs not already in the database.

With your help, the utility of the resource will further increase over time.

EDRC Deadlines Approaching

The 23rd European Drosophila Research Conference will be held in Barcelona this October. Deadlines are approaching for authors registration and early-bird participants.

CRISPRs in flies. Two breaking reports.

Bassett AR, Tibbit C, Ponting CP, Liu JL. Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System. Cell Rep. 2013 Jun 27. PMID: 23827738.

Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G. Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in Drosophila. Genetics. 2013 Jul 5. PMID: 23833182.

See also this post on an earlier CRISPR paper.

Genome-wide screen--Viral replication. Breaking report.

Hopkins KC, McLane LM, Maqbool T, Panda D, Gordesky-Gold B, Cherry S. A genome-wide RNAi screen reveals that mRNA decapping restricts bunyaviral replication by limiting the pools of Dcp2-accessible targets for cap-snatching. Genes Dev. 2013 PMID: 23824541.

TAL-based repression and activation in Drosophila

Crocker J, Stern DL. TALE-mediated modulation of transcriptional enhancers in vivo. Nat Methods. 2013 Jun 30. PMID: 23817068.

Synaptic development & maintenance. Recent report.

On my desk to read today? This report of fly RNAi interrogation of about 2000 genes.

Valakh V, Naylor SA, Berns DS, DiAntonio A. A large-scale RNAi screen identifies functional classes of genes shaping synaptic development and maintenance. Dev Biol. 2012 Jun 15;366(2):163-71. PMID: 22542760; PMCID: PMC3358632.

From the abstract:  "... genes with essential roles in non-neural tissues may be missed in traditional loss-of-function screens. In an effort to circumvent this limitation, we used neuron-specific RNAi knock down in Drosophila and assayed the formation, growth, and maintenance of the neuromuscular junction (NMJ). We examined 1970 Drosophila genes, each of which has a conserved ortholog in mammalian genomes. Knock down of 158 genes in post-mitotic neurons led to abnormalities in the neuromuscular system ..."

UP-TORR. A new tool from the DRSC. Find RNAi reagents--using today's information!

The DRSC has launched a new tool! 

And a corresponding paper is now out in the journal Genetics.

Hu Y, Roesel C, Flockhart I, Perkins L, Perrimon N, Mohr S. UP-TORR: Online Tool for Accurate and Up-to-Date Annotation of RNAi Reagents. Genetics. 2013 Jun 21. PMID: 23792952.

UP-TORR, named for "Updated Targets of RNAi Reagents" checks daily for new updates to fly gene annotations and RNAi reagent collections (including TRiP, NIG-Japan and VDRC RNAi fly stocks). This lets you view the most updated interpretation of the gene-to-RNAi-reagent relationship.

Why does this matter? Because gene annotations change--sometimes a lot--over time, changing the interpretation of on-targets and off-targets of a reagent.

Please check out our paper to learn more. We also have a help page and a video demo online. As always, feedback on the online tool is welcome.

DKFZ announces version 11 of GenomeRNAi

The DKFZ recently announced launch of a version 11 of their meta-database of fly and human RNAi screen results, GenomeRNAi. Read their news item here.

Morphological complexity. Large-scale fly cell RNAi screen. Breaking report.

Data corresponding to the screen reported in this paper are available at flyrnai.org and through PubChem. The fly cell screen was done in Kc cells. The study includes mammalian cell follow-up.

Yin et al. A screen for morphological complexity identifies regulators of switch-like transitions between discrete cell shapes. Nature Cell Biology (2013) doi:10.1038/ncb2764.

*See also* Olson MF. Finding the shape-shifter genes. Nat Cell Biol. 2013 PMID: 23817235.

piRNAs--genome-wide screen. Breaking report.

On my desk to read today? This report of an RNAi screen performed in ovarian somatic sheet cells.

Muerdter F, Guzzardo PM, Gillis J, Luo Y, Yu Y, Chen C, Fekete R, Hannon GJ. A Genome-wide RNAi Screen Draws a Genetic Framework for Transposon Control and Primary piRNA Biogenesis in Drosophila. Mol Cell. 2013 May 9. PMID: 23665228.

Differential RNAi and small libraries. Recent report.

Here's an example of a study that used two of the DRSC small libraries. These libraries have an optimal layout (limits edge effects) and average 2 or more unique dsRNAs per gene.

Garcia MA, Alvarez MS, Sailem H, Bousgouni V, Sero J, Bakal C. Differential RNAi screening provides insights into the rewiring of signalling networks during oxidative stress. Mol Biosyst. 2012 Oct;8(10):2605-13. PMID: 22790786.

New small library for cell-based RNAi screens: RNA binding proteins

For blog readers first: The DRSC recently created a new library for cell-based RNAi. The library targets RNA binding proteins. Average of 2 unique reagents per gene. Please contact the DRSC director for more info.

EDRC abstract deadline extended to June 12th

There's still to submit an abstract to the 23rd European Drosophila Research Conference. Just saw a notice that the abstract deadline was extended to June 12, 2013.

Catching up--RNAi screen for modifiers of disease-related toxicity

VoSSfeldt H, Butzlaff M, PrüSSing K, Ní Chárthaigh RA, Karsten P, Lankes A, Hamm S, Simons M, Adryan B, Schulz JB, Voigt A. Large-scale screen for modifiers of ataxin-3-derived polyglutamine-induced toxicity in Drosophila. PLoS One. 2012;7(11):e47452. PMID: 23139745; PMCID: PMC3489908.

Recent review--immune responses

This review appears to have a particular focus on the use of RNAi screening to study Drosophila immune responses.

Valanne S. Functional genomic analysis of the Drosophila immune response. Dev Comp Immunol. 2013 May 21. PMID: 23707784.

Breaking report--using mass spec to look at in vivo fly RNAi knockdown

Glukhova VA, Tomazela DM, Findlay GD, Monnat RJ, Maccoss MJ. Rapid assessment of  RNAi-mediated protein depletion by selected reaction monitoring (SRM) mass spectrometry. J Proteome Res. 2013 May 28. PMID: 23713831.

Recent review--RNAi and viral defense

Karlikow M, Goic B, Saleh MC. RNAi and antiviral defense in Drosophila: Setting up a systemic immune response. Dev Comp Immunol. 2013 May 14. PMID: 23684730.

CRISPR technology in flies

Of course this is not RNAi but it's the kind of technology we like to keep aware of here at the DRSC. So I thought I'd share it on the blog.

Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013 May 24. PMID: 23709638.

Breaking report--piRNA Pathway

These authors used an in vivo RNAi screen in the fly ovary to interrogate the piRNA pathway.

Handler D, Meixner K, Pizka M, Lauss K, Schmied C, Gruber FS, Brennecke J. The Genetic Makeup of the Drosophila piRNA Pathway. Mol Cell. 2013 May 9. PMID: 23665231

New disease-relevant cell-based assay--Yersinia enterocolitica

These authors took advantage of the growth temperature of Drosophila cultured cells to establish a cell model for the GI pathogen Y. enterocolitica.

Walker KA, Maltez V, Hall JD, Vitko NP, Miller VL. A Phenotype at Last: Essential Role for the Yersinia enterocolitica Ysa Type III Secretion System in a Drosophila S2 cell model. Infect Immun. 2013 Apr 29. PMID: 23630961.

Acentriolar cell lines. Recent report.

Lecland N, Debec A, Delmas A, Moutinho-Pereira S, Malmanche N, Bouissou A, Dupré C, Jourdan A, Raynaud-Messina B, Maiato H, Guichet A. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant. Biol Open. 2013 Mar 15;2(3):314-23. doi: 10.1242/bio.20133327. PubMed PMID: 23519377; PubMed Central PMCID: PMC3603413.

From the abstract: "pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly."

Flies & phosphate. Breaking report.

This paper includes report of a full-genome cell-based RNAi screen performed at the DRSC and in vivo RNAi follow-up.

Bergwitz C, Wee MJ, Sinha S, Huang J, Derobertis C, Mensah LB, Cohen J, Friedman A, Kulkarni M, Hu Y, Vinayagam A, Schnall-Levin M, Berger B, Perkins LA, Mohr SE, Perrimon N. Genetic determinants of phosphate response in Drosophila. PLoS One. 2013;8(3):e56753. PubMed PMID: 23520455; PubMed Central PMCID: PMC3592877.

Targeted in vivo double-RNAi screen. Breaking report.

Also on my desk to read today? This report of a targeted double-knockdown screen.

Marinho J, Martins T, Neto M, Casares F, Pereira PS. The nucleolar protein Vito/Nol12 is required for the growth and differentiation progression activities of the Dpp pathway during Drosophila eye development. Dev Biol. 2013 Feb 14. PubMed PMID: 23416177.

Fly RNAi pathway and viral defense. Breaking reports.

On my desk to read today? These papers on RNAi as a viral defense.

Sabin LR, Zheng Q, Thekkat P, Yang J, Hannon GJ, Gregory BD, Tudor M, Cherry S. Dicer-2 processes diverse viral RNA species. PLoS One. 2013;8(2):e55458. PMID: 23424633; PMCID: PMC3570552

From the abstract: "This study uncovered several novel, heavily targeted features within viral genomes, offering insight into viral replication, viral immune evasion strategies, and the mechanism of antiviral RNAi."

Goic B, Vodovar N, Mondotte JA, Monot C, Frangeul L, Blanc H, Gausson V, Vera-Otarola J, Cristofari G, Saleh MC. RNA-mediated interference and reverse transcription control the persistence of RNA viruses in the insect model Drosophila. Nat Immunol. 2013 Feb 24. doi: 10.1038/ni.2542. PubMed PMID: 23435119.

From the abstract: "Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence."

Meeting Announcement--European Drosophila Conference--October 2013

I recently saw this announcement: "The 23rd European Drosophila Research Conference will be held in Barcelona on the 16th-19th October 2013. We encourage all of you to join us in this special event and to share with us all the latest developments in the growing community of Drosophila research.The Congress website, www.edrc2013.org, will be constantly updated to keep you informed about all the latest news regarding the scientific programme."

COMPLEAT complex enrichment tool

The DRSC is pleased to announce that we are making COMPLEAT available in our suite of online software tools.

Conceptually similar to gene set enrichment, COMPLEAT makes it possible to look for enrichment of predicted protein complexes in a large-scale RNAi data set--including those with multiple conditions or time-points. You'll see a graph (left-hand side) and can click do draw a polygon around points on the graph to view those specific complexes (right-hand side).

The online tool web pages include a Demo (narrated video) and a set of Example Files so you can test out the tool and see the data upload format. As always, your feedback is welcome!

Here's the citation and abstract from the paper, as well as a screenshot.

Vinayagam A, Hu Y, Kulkarni M, Roesel C, Sopko R, Mohr SE, Perrimon N. Protein complex-based analysis framework for high-throughput data sets.  Sci Signal. 2013 Feb 26;6(264):rs5. PMID: 23443684

Abstract: Analysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal-regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.

Arthropod Genomics Symposium--Abstract deadline approaching

The abstract submission deadline for the 7th Annual Arthropod Genomics Symposium is approaching--March 1st is the due-date for posters to be considered for platform presentations. The event itself is scheduled for June 12-15, 2013 at the University of Notre Dame.

Androgen Receptor & Prostate Cancer. From fly RNAi screen to human cell follow-up. Breaking report.

On my desk to read this President's Day? This report of a genome-wide RNAi screen performed with the NYU RNAi Core using the DRSC 2.0 genome-wide library.

Imberg-Kazdan K, Ha S, Greenfield A, Poultney CS, Bonneau R, Logan SK, Garabedian MJ. A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells. Genome Res. 2013 Feb 12. PubMed PMID: 23403032.

Ranking the Rankings. Breaking report.

On my desk to read today--this report of comparison among large-scale screen ranking methods and development of a new approach.

Siebourg J, Merdes G, Misselwitz B, Hardt WD, Beerenwinkel N. Stability of gene rankings from RNAi screens. Bioinformatics. 28(12):1612-8. PMID: 22513992.

RNAi rescue assay and snRNAs. Breaking report.

Chen J, Waltenspiel B, Warren WD, Wagner EJ. Functional Analysis of the Integrator Subunit 12 Identifies a Microdomain that Mediates Activation of the Drosophila Integrator Complex. J Biol Chem. 2013 Jan 3. PubMed PMID: 23288851.

Image analysis script for stress granules and processing bodies. Breaking report.

These authors describe a script developed to analyze a specific set of phenotypes in high-throughput image-based screens such as RNAi screens.

Perez-Pepe M, Slomiansky V, Loschi M, Luchelli L, Neme M, Thomas MG, Boccaccio GL. BUHO: A MATLAB Script for the Study of Stress Granules and Processing Bodies by High-Throughput Image Analysis. PLoS One. 2012;7(12):e51495. doi: 10.1371/journal.pone.0051495. PMID: 23284702; PMCID: PMC3527446.

RNAi and RNA analysis. Cell cycle study. Breaking report.

These authors follow RNAi screening for DNA content in S2 cells with transcriptional profiling to uncover transcriptional networks. Bonke M, Turunen M, Sokolova M, Vähärautio A, Kivioja T, Taipale M, Björklund M, Taipale J. Transcriptional networks controlling the cell cycle. G3 (Bethesda). 2013 Jan;3(1):75-90. doi: 10.1534/g3.112.004283. PubMed PMID: 23316440.

Targeted in vivo screen for Notch pathway regulators. Breaking report.

Zhang J, Liu M, Su Y, Du J, Zhu AJ. A Targeted In Vivo RNAi Screen Reveals Deubiquitinases as New Regulators of Notch Signaling. G3 (Bethesda). 2012 Dec;2(12):1563-75. doi: 10.1534/g3.112.003780. PMID: 23275879; PMCID: PMC3516478.

S2 cell and RNAi system for exploring phagocytosis of L. donovani

Peltan A, Briggs L, Matthews G, Sweeney ST, Smith DF. Identification of Drosophila Gene Products Required for Phagocytosis of Leishmania donovani. PLoS One. 2012;7(12):e51831. doi: 10.1371/journal.pone.0051831. PMID: 23272175; PMCID: PMC3521716.

From the abstract:  "Utilising the phagocytic capability of Drosophila S2 cells, together with available tools for modulating gene expression by RNAi, we have developed an experimental system in which to identify host proteins of this type on a genome-wide scale ... These observations suggest that this experimental approach has the potential to identify a large number of host effectors required for efficient parasite uptake and maintenance.

New insights into the "germline-specific RNAi mechanism known as the Piwi-interacting RNA (piRNA) pathway"

Gell S L, Reenan RA. Mutations to the piRNA Pathway Component aubergine Enhance Meiotic Drive of Segregation Distorter in Drosophila melanogaster. Genetics. PMID: 23267055.