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

• Published in: G3 : genes - genomes - genetics 2016-08, Vol.6 (8), p.2467-2478
• Main Authors: 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
• Format: Article
• Language: English
• Subjects: Animals; Animals, Genetically Modified; Cell Nucleus - metabolism; CRISPR; CRISPR-Cas Systems; Drosophila; Drosophila - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Female; Germ Cells; Green Fluorescent Proteins - genetics; Investigations; loss-of-function; Mutation; Protein Isoforms - genetics; Repressor Proteins - genetics; Repressor Proteins - metabolism; RNA Interference; RNA, Guide, CRISPR-Cas Systems; stem cell; Stem Cells; Transcription Factors - genetics
• ISSN: 2160-1836; 2160-1836
• DOI: 10.1534/g3.116.028571

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.