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

G3 (Bethesda). 2016 Aug 9;6(8):2467-78. 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.

Keywords: CRISPR; Drosophila; loss-of-function; stem cell.

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems*
  • Cell Nucleus / metabolism
  • Drosophila / genetics*
  • Drosophila Proteins / genetics*
  • Drosophila Proteins / metabolism
  • Female
  • Germ Cells
  • Green Fluorescent Proteins / genetics
  • Mutation
  • Protein Isoforms / genetics
  • RNA Interference*
  • RNA, Guide
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism
  • Stem Cells
  • Transcription Factors / genetics*

Substances

  • Drosophila Proteins
  • MESR4 protein, Drosophila
  • Protein Isoforms
  • RNA, Guide
  • Repressor Proteins
  • Tif-IA protein, Drosophila
  • Transcription Factors
  • Green Fluorescent Proteins