A Combination of CRISPR/Cas9 and Standardized RNAi as a Versatile Platform for the Characterization of Gene Function.
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.
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
Sebastian
S
Wissel
Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany.
Anja
A
Kieser
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
Tetsuo
T
Yasugi
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
Peter
P
Duchek
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria Institute of Molecular Pathology, 1030 Vienna, Austria.
Elisabeth
E
Roitinger
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
Joseph
J
Gokcezade
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
Victoria
V
Steinmann
Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany.
Ulrike
U
Gaul
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria Institute of Molecular Pathology, 1030 Vienna, Austria.
Karl
K
Mechtler
Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany.
Klaus
K
Förstemann
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria juergen.knoblich@imba.oeaw.ac.at ralph.neumueller@boehringer-ingelheim.com.
Jürgen A
0000-0002-6751-3404
JA
Knoblich
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany juergen.knoblich@imba.oeaw.ac.at ralph.neumueller@boehringer-ingelheim.com.
Ralph A
RA
Neumüller
D000818:Animals; D030801:Animals, Genetically Modified; D064113:CRISPR-Cas Systems; D002467:Cell Nucleus / Q000378:metabolism; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005854:Germ Cells; D049452:Green Fluorescent Proteins / Q000235:genetics; D009154:Mutation; D020033:Protein Isoforms / Q000235:genetics; D034622:RNA Interference; D000094704:RNA, Guide, CRISPR-Cas Systems; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013234:Stem Cells; D014157:Transcription Factors / Q000235:genetics*
D029721:Drosophila Proteins; C000601237:MESR4 protein, Drosophila; D020033:Protein Isoforms; D000094704:RNA, Guide, CRISPR-Cas Systems; D012097:Repressor Proteins; C526066:Tif-IA protein, Drosophila; D014157:Transcription Factors; D049452:Green Fluorescent Proteins
CRISPR; Drosophila; loss-of-function; stem cell