CRISPR, gene drives and safety

Two recent papers speak to CRISPR, gene drives and safety:



Akbari et al. 2015 Safeguarding gene drive experiments in the laboratory. in Early online in Policy Forum section of Science.  

 Abstract: "Multiple strategies are needed to ensure safe gene drive experiments."

Unckless et al. 2015 Modeling the Manipulation of Natural Populations by the Mutagenic Chain Reaction Early online at Genetics.

From the Discussion section:  "... there are conditions in which accidental introductions of a single individual can lead to fixation of the MCR allele ..."

Beyond fruit flies--iBeetle large-scale RNAi screen in Tribolium

Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G. The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun. 2015 Jul 28;6:7822. PMID: 26215380.

From the abstract: "Genetic screens are powerful tools to identify the genes required for a given biological process. .. for technical reasons, comprehensive screens have been restricted to very few model organisms. ... although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum ... This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila."

Don't miss: "New fields of research" section of the article, which describes studies, such as of the 'odiferous stink glands,' that can be done in Tribolium but not Drosophila.

Glia-specific RNAi screen explores circadian behavior

Ng FS, Jackson FR. The ROP vesicle release factor is required in adult Drosophila glia for normal circadian behavior. Front Cell Neurosci. 2015 Jul 3;9:256. PMID: 26190976.

Drosophila in vivo RNAi screen explores ovarian follicle cell differentiation

Jia D, Soylemez M, Calvin G, Bornmann R, Bryant J, Hanna C, Huang YC, Deng WM. A large-scale in vivo RNAi screen to identify genes involved in Notch-mediated follicle cell differentiation and cell cycle switches. Sci Rep. 2015 Jul 24;5:12328. PMID: 26205122.

Drosophila CRISPR library reported by Bassett et al

Bassett AR, Kong L, Liu JL. A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells. J Genet Genomics. 2015 Jun 20;42(6):301-9. PMID: 26165496.

DRSC publishes RBP library resource including 'baseline data' available lots of ways--including interactive!

The DRSC is pleased to announce our new cell-based RNAi library targeting RNA Binding Proteins and corresponding 'baseline' data set. These data should be a helpful reference for anyone analyzing data from screens of the same library with more sophisticated assays.  

Big thanks to all the folks who worked hard to make this possible!

Mohr, Hu, Rudd, Buckner, Gilly, Foster, Sierzputowska, Comjean, Ye and Perrimon (2015) Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells. Early online at G3 July 2015.

At a DRSC webpage we summarize ways to view the data.

Just one example--when you view the total ATP readout data at Plot.ly you can hover to see gene and reagent identifiers, zoom in on sub-sections of the graph, etc. The data are also available at the DRSC, at NCBI PubChem and no doubt will be imported by the folks at GenomeRNAi.

New report on TALEN gene editing in flies

An optimized TALEN application for mutagenesis and screening in Drosophila melanogaster. http://t.co/zcvHP87UWl

— flypapers (@fly_papers) July 22, 2015

S2 cell-based assay related to bioactive lipids

Wang P, Wang Q, Yang L, Qin QL, Wu YJ. Characterization of lysophosphatidylcholine-induced changes of intracellular calcium in Drosophila S2 cells. Life Sci. 2015 Jun 15;131:57-62. PMID: 25736969.

From the abstract: "Lysophosphatidylcholine (LPC), a bioactive lipid, regulates a wide array of biological processes. ... We aimed to study the mechanism of LPC-induced [Ca(2+)]i changes in Drosophila S2 cells. ... The [Ca(2+)]i of Drosophila S2 cells was measured by fluorescence spectrophotometry after loading the cells with the calcium-sensitive fluorescent probe Fura-2/AM. ...
Our results demonstrated that LPC could cause a rapid, dose-dependent increase in the [Ca(2+)]i in the presence of external calcium ([Ca(2+)]e). ... The LPC-induced [Ca(2+)]i increase in S2 cells characterized in this study may shed light on the study of NTE/SWS protein function in general because the enzyme is responsible for the deacylation of LPC."

in vivo RNAi contributes to follow-up of GWAS candidate aggression-related genes

Shorter J, Couch C, Huang W, Carbone MA, Peiffer J, Anholt RR, Mackay TF. Genetic architecture of natural variation in Drosophila melanogaster aggressive behavior. Proc Natl Acad Sci U S A. 2015 Jul 7;112(27):E3555-63. PMID: 26100892.

From the abstract: "Aggression is an evolutionarily conserved complex behavior ... Here, we performed genome-wide association analyses using the inbred, sequenced lines of the Drosophila melanogaster Genetic Reference Panel (DGRP) and replicate advanced intercross populations derived from the most and least aggressive DGRP lines. We identified genes that have been previously implicated in aggressive behavior as well as many novel loci ... We used mutations and RNAi knock-down alleles to functionally validate 79% of the candidate genes and 75% of the candidate epistatic interactions tested. Epistasis for aggressive behavior causes cryptic genetic variation in the DGRP that is revealed by changing allele frequencies in the outbred populations derived from extreme DGRP lines. This phenomenon may pertain to other fitness traits and species, with implications for evolution, applied breeding, and human genetics."

Report explores mechanisms underlying target cleavage by RISC

Yao C, Sasaki HM, Ueda T, Tomari Y, Tadakuma H. Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex. Mol Cell. 2015 Jul 2;59(1):125-32. PMID: 26140368.

From the abstract: "Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5' cleavage fragment from the guide 3' supplementary region first and then the 3' fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. ... "

Fly cell screen leads to findings relevant to cancer treatment (and its financial impact)

Thomas S, Fisher KH, Snowden JA, Danson SJ, Brown S, Zeidler MP. Methotrexate Is a JAK/STAT Pathway Inhibitor. PLoS One. 2015 Jul 1;10(7):e0130078. PMID: 26131691.

From the abstract: "... The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. ...We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. ... While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential."

Protocol paper -- qPCR to detect cell death-related transcripts following dsRNA treatment of fly cells

Denton D, Kumar S. Analyzing the Response of RNAi-Treated Drosophila Cells to Death Stimuli by Quantitative Real-Time Polymerase Chain Reaction. Cold Spring Harb Protoc. 2015 Jul 1;2015(7):pdb.prot086223. PMID: 26134907.

From the abstract: "A useful complement to animal studies is the use of Drosophila cell lines to analyze cell-death responses. ... Drosophila cell lines are very amenable to knockdown studies ... the cell lines are useful for investigating the response to death stimuli, following gene knockdown, by examining the expression of cell-death genes. This protocol describes the synthesis of dsRNA for treatment of Drosophila cells and the subsequent analysis of cell-death gene expression by quantitative real-time polymerase chain reaction (qPCR)."

Methods paper--RNAi screening and the centrosome

Dobbelaere J. Genome-wide RNAi screens in S2 cells to identify centrosome components. Methods Cell Biol. 2015;129:279-300.PMID: 26175444.

From the abstract: "... Genome-wide RNA interference (RNAi) allows for comprehensive screening ... Drosophila tissue culture cells provide an attractive model for such screens. ... Drosophila centrosomes are similar to their human counterparts, but less complex. Thus, all major centrosome components are conserved and fewer redundancies apply ... In this paper, we present detailed instructions for designing, performing, and analyzing a genome-wide screen in Drosophila tissue culture cells to identify centrosome components using a microscopy-based approach."

Fly gene list resource live and published

The DRSC is pleased to say that our GLAD gene list annotation database is live at flyrnai.org/glad and published in the Journal of Genomics!

The tool lets you view curated gene lists, corresponding information, and more. Improvable through community input on genes to add, publications to check out, etc. relevant to specific litst.