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G3 (Bethesda). 2016 Aug 9;6(8):2467-78. doi: 10.1534/g3.116.028571.

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

Author information

1
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria.
2
Gene Center, Ludwig-Maximilians-University Munich, 81377, Germany.
3
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria Institute of Molecular Pathology, 1030 Vienna, Austria.
4
Institute of Molecular Biotechnology Austria, 1030 Vienna, Austria juergen.knoblich@imba.oeaw.ac.at ralph.neumueller@boehringer-ingelheim.com.
5
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.

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.

KEYWORDS:

CRISPR; Drosophila; loss-of-function; stem cell

PMID:
27280787
PMCID:
PMC4978900
DOI:
10.1534/g3.116.028571
[Indexed for MEDLINE]
Free PMC Article

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