학술논문
An expanded auxin-inducible degron toolkit for Caenorhabditis elegans
Document Type
article
Author
Ashley, Guinevere E; Duong, Tam; Levenson, Max T; Martinez, Michael AQ; Johnson, Londen C; Hibshman, Jonathan D; Saeger, Hannah N; Palmisano, Nicholas J; Doonan, Ryan; Martinez-Mendez, Raquel; Davidson, Brittany R; Zhang, Wan; Ragle, James Matthew; Medwig-Kinney, Taylor N; Sirota, Sydney S; Goldstein, Bob; Matus, David Q; Dickinson, Daniel J; Reiner, David J; Ward, Jordan D
Source
Genetics. 217(3)
Subject
Language
Abstract
The auxin-inducible degron (AID) system has emerged as a powerful tool to conditionally deplete proteins in a range of organisms and cell types. Here, we describe a toolkit to augment the use of the AID system in Caenorhabditis elegans. We have generated a set of single-copy, tissue-specific (germline, intestine, neuron, muscle, pharynx, hypodermis, seam cell, anchor cell) and pan-somatic TIR1-expressing strains carrying a co-expressed blue fluorescent reporter to enable use of both red and green channels in experiments. These transgenes are inserted into commonly used, well-characterized genetic loci. We confirmed that our TIR1-expressing strains produce the expected depletion phenotype for several nuclear and cytoplasmic AID-tagged endogenous substrates. We have also constructed a set of plasmids for constructing repair templates to generate fluorescent protein::AID fusions through CRISPR/Cas9-mediated genome editing. These plasmids are compatible with commonly used genome editing approaches in the C. elegans community (Gibson or SapTrap assembly of plasmid repair templates or PCR-derived linear repair templates). Together these reagents will complement existing TIR1 strains and facilitate rapid and high-throughput fluorescent protein::AID tagging of genes. This battery of new TIR1-expressing strains and modular, efficient cloning vectors serves as a platform for straightforward assembly of CRISPR/Cas9 repair templates for conditional protein depletion.