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      A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function

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          Abstract

          Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila. miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a , and the previously uncharacterized gene MESR4 , in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.

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          Most cited references 29

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          Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.

           Yu Luo,  Belinda Lee (2001)
          We have modified an FLP/FRT-based genetic mosaic system to label either neurons derived from a common progenitor or isolated single neurons, in the Drosophila CNS. These uniquely labeled neurons can also be made homozygous for a mutation of interest within an otherwise phenotypically wild-type brain. Using this new mosaic system, not only can normal brain development be described with unprecedented single cell resolution, but also the underlying molecular mechanisms can be investigated by identifying genes that are required for these developmental processes.
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            Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes.

            A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.
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              RNAi screening comes of age: improved techniques and complementary approaches.

              Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                7 June 2016
                August 2016
                : 6
                : 8
                : 2467-2478
                Affiliations
                [* ]Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria
                []Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany
                []Institute of Molecular Pathology, 1030 Vienna, Austria
                Author notes
                [1]

                These authors contributed equally to this work.

                [2]

                Present address: Mathematical Neuroscience Research Unit, Institute for Frontier Science Initiative, Kanazawa University, Ishikawa 920-8640, Japan.

                [3 ]Corresponding authors: Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria. E-mail: juergen.knoblich@ 123456imba.oeaw.ac.at ; and Boehringer Ingelheim RCV, Doktor-Boehringer-Gasse 5-11, 1120 Vienna, Austria. E-mail: ralph.neumueller@ 123456boehringer-ingelheim.com
                [4]

                Present address: Boehringer Ingelheim RCV Oncology, 1120 Vienna, Austria.

                Article
                GGG_028571
                10.1534/g3.116.028571
                4978900
                27280787
                Copyright © 2016 Wissel et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, Tables: 0, Equations: 0, References: 37, Pages: 12
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                Investigations

                Genetics

                crispr, stem cell, drosophila, loss-of-function

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