Monday, March 30, 2015

NIH NIGMS announces new strategic plan

Many readers will be interested to learn that NIGMS at NIH has released a new strategic plan. An announcement, including a link to a PDF copy of the plan, is at the NIGMS Feedback Loop blog.

Report of 'systems-level interrogation' includes DRSC RNAi screen data, phosphoproteomics and in vivo analyses

Sopko R, Lin YB, Makhijani K, Alexander B, Perrimon N, Br├╝ckner K. A systems-level interrogation identifies regulators of Drosophila blood cell number and survival. PLoS Genet. 2015 Mar 6;11(3):e1005056. PMID: 25749252; PMCID: PMC4352040.

From the abstract: "Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. ... Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and ... Ecdysone receptor (EcR) and ultraspiracle (usp) ... as Pvr Suppressors. ... Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. ... our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems."

Friday, March 27, 2015

GenomeRNAi beta-testing a 'screen comparison tool'

I recently stumbled across this beta-test at GenomeRNAi of a screen comparison tool. Enter one or a list of genes and view, heat-map style, in what screens those genes were hits. They indicate they're interested to receive feedback. The default option is human genes but Drosophila also supported.

Wednesday, March 25, 2015

"Trojan exon" variation on the theme of MiMIC and CRISPR insertion strategies

Diao F, Ironfield H, Luan H, Diao F, Shropshire WC, Ewer J, Marr E, Potter CJ, Landgraf M, White BH. Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes. Cell Rep. 2015 Mar 3;10(8):1410-21. PMID: 25732830.

From the abstract: "... we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes ... Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest ... We demonstrate the efficacy of this approach in Drosophila ... We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system."

Protocol report--dual luciferase assays

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; PMCID: PMC4059354.

Friday, March 20, 2015

The cut that keeps on cutting: CRISPR-Cas mutagenic chain reaction approach

Gantz and Bier (2015) The mutagenic chain reaction: A method for converting heterozygous to homozygous mutations. Science early online DOI: 10.1126/science.aaa5945


Abstract: "An organism with a single recessive loss-of-function allele will typically have a wild-type phenotype while individuals homozygous for two copies of the allele will display a mutant phenotype. Here, we develop a method that we refer to as the mutagenic chain reaction (MCR), which is based on the CRISPR/Cas9 genome editing system for generating autocatalytic mutations to generate homozygous loss-of-function mutations. We demonstrate in Drosophila that MCR mutations efficiently spread from their chromosome of origin to the homologous chromosome thereby converting heterozygous mutations to homozygosity in the vast majority of somatic and germline cells. MCR technology should have broad applications in diverse organisms."

See also Science magazine's news coverage of the report. 

Wednesday, March 18, 2015

CRISPR off-targets study in human cells

Findings relevant to fly studies? Will be interesting to see.

Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015 Mar;12(3):237-43. PMID: 25664545.

From the abstract: "... genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. ... We also showed that Cas9 nucleases can be highly specific ... and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. ..."