Monday, August 22, 2016

Using the white gene as an indicator of CRISPR-based editing

Ge DT, Tipping C, Brodsky MH, Zamore PD. Rapid Screening for CRISPR-Directed Editing of the Drosophila Genome Using white Co-Conversion. G3 (Bethesda). 2016 Aug 19. pii: g3.116.032557. PMID: 27543296.

From the abstract: "... Here, we describe a strategy that reduces the time and effort required to identify flies with targeted genomic changes. The strategy uses editing of the white gene, evidenced by altered eye color, to predict successful editing of an unrelated gene-of-interest. The red eyes of wild-type flies are readily distinguished from white-eyed (end joining-mediated loss of White function) or brown-eyed (recombination-mediated conversion to the whitecoffee allele) mutant flies. ... We find that end joining-mediated mutations often show signatures of microhomology-mediated repair and that recombination-based mutations frequently involve donor plasmid integration at the target locus. Finally, we show that gap repair induced by two guide RNAs more reliably converts the intervening target sequence, whereas the use of Lig4169 mutants to suppress end joining does not improve recombination efficacy."

Genome-wide screen in study of heterochromatin factor HP1a

The cell-based RNAi screen described in this study was done using a DRSC library and follow-up reagents. Congratulations, Joel and colleagues!

Swenson JM, Colmenares SU, Strom AR, Costes SV, Karpen GH. The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. Elife. 2016 Aug 11;5. pii: e16096. PMID: 27514026; PMCID: PMC4981497.

Abstract: "Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions."

Friday, August 5, 2016

Genome-wide in vivo screen looks at male germline stem cells

Liu Y, Ge Q, Chan B, Liu H, Singh SR, Manley J, Lee J, Weideman AM, Hou G, Hou SX. Whole-animal genome-wide RNAi screen identifies networks regulating male germline stem cells in Drosophila. Nat Commun. 2016 Aug 3;7:12149. PMID: 27484291.

From the abstract: "... To identify genes and pathways that regulate stem-cell fates in the whole organism, we perform a genome-wide transgenic RNAi screen through ubiquitous gene knockdowns, focusing on regulators of adult Drosophila testis germline stem cells (GSCs). Here we identify 530 genes that regulate GSC maintenance and differentiation. Of these, we further knock down 113 selected genes using cell-type-specific Gal4s and find that more than half were external regulators, that is, from the local microenvironment or more distal sources. ..."

Talk of transvection, RNAi, and Gal4-UAS, all in one G3 report

Tuesday, July 5, 2016

DRSC & TRiP website -- All new!

We just went live with a completely redone DRSC & TRiP website.

And a new name, DRSC/TRiP Functional Genomics Resources.

The flyrnai.org pages will re-direct (except for online tools, which will still live at that URL).

Or you can check it out directly at http://fgr.hms.harvard.edu/

Feedback welcome!

Thursday, June 30, 2016

Gene editing in fly cells--new report from Kunzelmann et al. in G3

Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells. G3 (Bethesda). 2016 Jun 1;6(6):1777-85. PMID: 27172193

From the abstract: "... Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. ..."

EP-MS2 method and RNAi follow-up used in screen for localized RNAs

Misra M, Edmund H, Ennis D, Schlueter MA, Marot JE, Tambasco J, Barlow I, Sigurbjornsdottir S, Mathew R, Vallés AM, Davis I, Leptin M, Gavis ER. A Genome-Wide Screen for Dendritically Localized RNAs Identifies Genes Required for Dendrite Morphogenesis. G3 (Bethesda). 2016 Jun 3. pii: g3.116.030353. PMID: 27260999.

From the abstract: "Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains ... However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. ..."