Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins

Melinda Bence, Ferenc Jankovics, Tamás Lukácsovich, M. Erdélyi

Research output: Contribution to journalEditorial

9 Citations (Scopus)

Abstract

Inducible protein degradation techniques have considerable advantages over classical genetic approaches, which generate loss-of-function phenotypes at the gene or mRNA level. The plant-derived auxin-inducible degradation system (AID) is a promising technique which enables the degradation of target proteins tagged with the AID motif in nonplant cells. Here, we present a detailed characterization of this method employed during the adult oogenesis of Drosophila. Furthermore, with the help of CRISPR/Cas9-based genome editing, we improve the utility of the AID system in the conditional elimination of endogenously expressed proteins. We demonstrate that the AID system induces efficient and reversible protein depletion of maternally provided proteins both in the ovary and the early embryo. Moreover, the AID system provides a fine spatiotemporal control of protein degradation and allows for the generation of different levels of protein knockdown in a well-regulated manner. These features of the AID system enable the unraveling of the discrete phenotypes of genes with highly complex functions. We utilized this system to generate a conditional loss-of-function allele which allows for the specific degradation of the Vasa protein without affecting its alternative splice variant (solo) and the vasa intronic gene (vig). With the help of this special allele, we demonstrate that dramatic decrease of Vasa protein in the vitellarium does not influence the completion of oogenesis as well as the establishment of proper anteroposterior and dorsoventral polarity in the developing oocyte. Our study suggests that both the localization and the translation of gurken mRNA in the vitellarium is independent from Vasa.

Original languageEnglish
Pages (from-to)1056-1069
Number of pages14
JournalFEBS Journal
Volume284
Issue number7
DOIs
Publication statusPublished - Apr 1 2017

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Drosophila Proteins
Indoleacetic Acids
Genes
Degradation
Proteolysis
Oogenesis
Proteins
Alleles
Phenotype
Protein Biosynthesis
Drosophila
Oocytes
Gene Editing
Ovary
Embryonic Structures
Messenger RNA

Keywords

  • auxin-inducible degradation
  • CRISPR/Cas9
  • Drosophila melanogaster
  • induced protein depletion
  • Vasa

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins. / Bence, Melinda; Jankovics, Ferenc; Lukácsovich, Tamás; Erdélyi, M.

In: FEBS Journal, Vol. 284, No. 7, 01.04.2017, p. 1056-1069.

Research output: Contribution to journalEditorial

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