Journal
GENETICS
Volume 217, Issue 3, Pages -Publisher
GENETICS SOCIETY AMERICA
DOI: 10.1093/genetics/iyab006
Keywords
C. elegans; AID system; SapTrap; self-excising cassette; CRISPR/Cas9; Transport Inhibitor Response 1
Categories
Funding
- NIH/NIGMS [R00 GM107345, R01 GM138701]
- National Science Foundation (NSF) Division of Molecular and Cellular Biosciences (CAREER award) [1942922]
- NIH NIGMS [R01 GM121597]
- Damon Runyon Cancer Research Foundation [DRR-47-17]
- NIGMS [R01 GM121597-02S2]
- American Cancer Society [132969-PF-18-22601-CSM]
- NICHD [F31 HD100091]
- Cancer Prevention and Research Institute of Texas Grant [RR170054]
- NIH Office of Research Infrastructure Programs [P40 OD010440]
- [F32 GM131577]
- [R35 GM134838]
- [R00-GM115964]
- [R01 GM121625]
- Direct For Biological Sciences [1942922] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1942922] Funding Source: National Science Foundation
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This study presents a toolkit for enhancing the application of the AID system in Caenorhabditis elegans, including tissue-specific and pan-somatic TIR1-expressing strains, as well as plasmids for constructing repair templates for generating fluorescent protein::AID fusions. These reagents will complement existing TIR1 strains and facilitate rapid and high-throughput tagging of genes with fluorescent protein::AID.
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.
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