Friday, April 10, 2015
Transcription factor validated in follow-up to genome-wide RNAi screen related to viral-host cell interactions
Panda D, Gold B, Tartell MA, Rausch K, Casas-Tinto S, Cherry S. The Transcription Factor FoxK Participates with Nup98 To Regulate Antiviral Gene Expression. MBio. 2015 Apr 7;6(2). PMID: 25852164.
in vivo screen of kinases and phosphatases identifies Wnt signal regulators
Swarup S, Pradhan-Sundd T, Verheyen EM. Genome-wide identification of phospho-regulators of Wnt signaling in Drosophila. Development. 2015 Apr 15;142(8):1502-15. PMID: 25852200.
From the abstract: "... In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway ... Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. ... "
From the abstract: "... In this study, we performed a genome-wide in vivo RNAi screen for kinases and phosphatases that regulate the Wnt pathway under physiological conditions in the Drosophila wing disc. Our analyses have identified 54 high-confidence kinases and phosphatases capable of modulating the Wnt pathway, including 22 novel regulators. These candidates were also assayed for a role in the Notch pathway ... Additionally, each regulator of the Wnt pathway was evaluated in the wing disc for its ability to affect the mechanistically similar Hedgehog pathway. ... "
Wednesday, April 8, 2015
Report of improved CRISPR-based transcriptional activation approach includes work in Drosophila cultured cells
Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM. Highly efficient Cas9-mediated transcriptional programming. Nat Methods. 2015 Apr;12(4):326-8. PMID: 25730490.
From the abstract: "... modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. ..."
Drosophila S2R+ cultured cells are among the systems in which this approach was successfully tested. See Results and Supplementary Fig. 9.
From the abstract: "... modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. ..."
Drosophila S2R+ cultured cells are among the systems in which this approach was successfully tested. See Results and Supplementary Fig. 9.
Drosophila Models of Human Disease: Mammalian screen with fly follow-up identifies pot...
Drosophila Models of Human Disease: Mammalian screen with fly follow-up identifies pot...: Jimenez-Sanchez M, Lam W, Hannus M, Sönnichsen B, Imarisio S, Fleming A, Tarditi A, Menzies F, Ed Dami T, Xu C, Gonzalez-Couto E, Lazzeroni ...
C. elegans report presents simple rule for effective CRISPR sgRNA design
Farboud B, Meyer BJ. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design. Genetics. 2015 Feb 18. PMID: 25695951.
From the abstract: "... Here we describe and validate a strategy for Caenorhabditis elegans ... The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. ..."
Will this prove true in Drosophila as well? There are certainly guide designs reported to be effective in flies that break the GG rule. And it is hard to believe that the design algorithms developed by various groups would not have picked this up. Nevertheless, it's tempting to give it a try.
From the abstract: "... Here we describe and validate a strategy for Caenorhabditis elegans ... The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. ..."
Will this prove true in Drosophila as well? There are certainly guide designs reported to be effective in flies that break the GG rule. And it is hard to believe that the design algorithms developed by various groups would not have picked this up. Nevertheless, it's tempting to give it a try.
Monday, April 6, 2015
Efficient fly CRISPR with limited risk? Preprint from Port and colleagues compares sgRNA transgenic and MCR approaches
Interested to efficiently generate knockout flies without the potential risks presented by the mutagenic chain reaction (MCR) approach? Check out a new pre-print from Port et al.
Port, Muschalik and Bullock (preprint) CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research.
From the abstract: "... Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk."
Port, Muschalik and Bullock (preprint) CRISPR with independent transgenes is a safe and robust alternative to autonomous gene drives in basic research.
From the abstract: "... Here, we systematically evaluate available CRISPR/Cas reagents and experimental designs in Drosophila. Our findings allow evidence-based choices of Cas9 sources and strategies for generating knock-in alleles. We perform gene editing at a large number of target sites using a highly active Cas9 line and a collection of transgenic gRNA strains. The vast majority of target sites can be mutated with remarkable efficiency using these tools. We contrast our method to recently developed autonomous gene drive technology for genome engineering (Gantz & Bier, 2015) and conclude that optimised CRISPR with independent transgenes is as efficient, more versatile and does not represent a biosafety risk."
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