• Media type: E-Article
  • Title: A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function
  • Contributor: Wissel, Sebastian; Kieser, Anja; Yasugi, Tetsuo; Duchek, Peter; Roitinger, Elisabeth; Gokcezade, Joseph; Steinmann, Victoria; Gaul, Ulrike; Mechtler, Karl; Förstemann, Klaus; Knoblich, Jürgen A; Neumüller, Ralph A
  • imprint: Oxford University Press (OUP), 2016
  • Published in: G3 Genes|Genomes|Genetics
  • Language: English
  • DOI: 10.1534/g3.116.028571
  • ISSN: 2160-1836
  • Keywords: Genetics (clinical) ; Genetics ; Molecular Biology
  • Origination:
  • Footnote:
  • Description: <jats:title>Abstract</jats:title><jats:p>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.</jats:p>
  • Access State: Open Access