An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generatio

Bivalency in Drosophila embryos is associated with strong inducibility of Polycomb target genes

Polycomb group (PcG) and Trithorax group (TrxG) proteins orchestrate development of a multicellular organism by faithfully maintaining cell fate decisions made early in embryogenesis. An important chromatin mark connected to PcG/TrxG regulation is bivalent domains, the simultaneous presence of H3K27me3 and H3K4me3 on a given locus, originally identified in mammalian embryonic stem cells but considered to be absent in invertebrates. Here, we provide evidence for the existence of bivalency in fly embryos. Using a recently described PcG reporter fly line, we observed a strong reporter inducibility in the embryo and its sharp decrease in larval and adult stages. Analysis of the chromatin landscape of the reporter revealed a strong signal for the repressive PcG mark, H3K27me3, in all three developmental stages and, surprisingly, a strong signal for a transcriptionally activating H3K4me3 mark in the embryo. Using re-chromatin immunoprecipitation experiments, bivalent domains were also uncovered at endogenous PcG targets like the Hox genes

Single vector non-leaky gene expression system for Drosophila melanogaster

An ideal transgenic gene expression system is inducible, non-leaky, and well tolerated by the target organism. While the former has been satisfactorily realized, leakiness and heavy physiological burden imposed by the existing systems are still prominent hurdles in their successful implementation. Here we describe a new system for non-leaky expression of transgenes in Drosophila. PRExpress is based on a single transgenic construct built from endogenous components, the inducible hsp70 promoter and a multimerized copy of a Polycomb response element (PRE) controlled by epigenetic chromatin regulators of the Polycomb group. We show that this system is non-leaky, rapidly and strongly inducible, and reversible. To make the application of PRExpress user-friendly, we deliver the construct via site-specific integration.ISSN:2045-232

An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.ISSN:1362-4962ISSN:0301-561

dCas9-mediated dysregulation of gene expression in human induced pluripotent stem cells during primitive streak differentiation

CRISPR-based systems have fundamentally transformed our ability to study and manipulate stem cells. We explored the possibility of using catalytically dead Cas9 (dCas9) from S. pyogenes as a platform for targeted epigenetic editing in stem cells to enhance the expression of the eomesodermin gene (EOMES) during differentiation. We observed, however, that the dCas9 protein itself exerts a potential non-specific effect in hiPSCs, affecting the cell's phenotype and gene expression patterns during subsequent directed differentiation. We show that this effect is specific to the condition when cells are cultured in medium that does not actively maintain the pluripotency network, and that the sgRNA-free apo-dCas9 protein itself influences endogenous gene expression. Transcriptomics analysis revealed that a significant number of genes involved in developmental processes and various other genes with non-overlapping biological functions are affected by dCas9 overexpression. This suggests a potential adverse phenotypic effect of dCas9 itself in hiPSCs, which could have implications for when and how CRISPR/Cas9-based tools can be used reliably and safely in pluripotent stem cells.ISSN:1096-7176ISSN:1096-718