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

• Published in: The FEBS journal Vol. 284; no. 7; pp. 1056 - 1069
• Main Authors: Bence, Melinda, Jankovics, Ferenc, Lukácsovich, Tamás, Erdélyi, Miklós
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.04.2017
• Subjects: Alleles; Alternative splicing; Animals; auxin‐inducible degradation; Biodegradation; Cell Polarity - drug effects; Cell Polarity - genetics; CRISPR; CRISPR-Cas Systems; CRISPR/Cas9; DEAD-box RNA Helicases - deficiency; DEAD-box RNA Helicases - genetics; Degradation; Depletion; Dose-Response Relationship, Drug; Drosophila; Drosophila melanogaster; Drosophila melanogaster - cytology; Drosophila melanogaster - drug effects; Drosophila melanogaster - genetics; Drosophila melanogaster - growth & development; Drosophila Proteins - deficiency; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Editing; Embryo, Nonmammalian; Embryos; Female; Gene Editing - methods; Gene Expression Regulation, Developmental; Genomes; Genotype; Indoleacetic Acids - pharmacology; induced protein depletion; Insects; Levels; Localization; Maternal Inheritance; mRNA; Oocytes - cytology; Oocytes - drug effects; Oocytes - growth & development; Oocytes - metabolism; Oogenesis; Oogenesis - drug effects; Oogenesis - genetics; Ovary - cytology; Ovary - drug effects; Ovary - growth & development; Ovary - metabolism; Phenotype; Phenotypes; Polarity; Protein Biosynthesis; Protein Isoforms - genetics; Protein Isoforms - metabolism; Proteins; Proteolysis - drug effects; Transforming Growth Factor alpha - genetics; Transforming Growth Factor alpha - metabolism; Vasa; Vasa protein; Vasa; induced protein depletion; CRISPR/Cas9; Drosophila melanogaster; auxin-inducible degradation
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.14042

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. The plant‐derived auxin‐inducible degradation (AID) system permits the selective polyubiquitination and proteasomal degradation of target proteins tagged with the AID motif. First applied in yeast and cultured cells, it was recently shown to be effective in multicellular organisms, including somatic cells in Drosophila larval stages. Bence and colleagues now report that the AID system can be used to elicit rapid and reversible degradation of two maternal effect proteins, Nanos and Vasa, in the Drosophila ovary and early embryo. In addition, they combine the AID system with CRISPR/Cas9‐based genome‐editing techniques to eliminate endogenous proteins.