title: Combining the auxin-inducible degradation system with CRISPR/Cas9-based genome editing for the conditional depletion of endogenous Drosophila melanogaster proteins.
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.
authors.affiliation: Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Melinda
authors.initials: M
authors.lastname: Bence
authors.affiliation: Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Jankovics
authors.affiliation: Brain Research Institute, University of Zürich, Switzerland.
authors.forename: Tamás
authors.initials: T
authors.lastname: Lukácsovich
authors.affiliation: Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary.
authors.forename: Miklós
authors.initials: M
authors.lastname: Erdélyi
mesh_terms: D000483:Alleles; D000818:Animals; D064113:CRISPR-Cas Systems; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D004625:Embryo, Nonmammalian; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D005838:Genotype; D007210:Indoleacetic Acids / Q000494:pharmacology*; D000072741:Maternal Inheritance; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D009866:Oogenesis / Q000187:drug effects / Q000235:genetics; D010053:Ovary / Q000166:cytology / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D010641:Phenotype; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D016211:Transforming Growth Factor alpha / Q000235:genetics* / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D007210:Indoleacetic Acids; D020033:Protein Isoforms; D016211:Transforming Growth Factor alpha; C083730:grk protein, Drosophila; C088269:vas protein, Drosophila; D053487:DEAD-box RNA Helicases
keywords: 
  Drosophila melanogaster
  ; CRISPR/Cas9; Vasa; auxin-inducible degradation; induced protein depletion