An expanded auxin-inducible degron toolkit for Caenorhabditis elegans

Genetics. 2021 Mar 31;217(3):iyab006. doi: 10.1093/genetics/iyab006.

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.

Keywords: C. elegans; AID system; CRISPR/Cas9; SapTrap; Transport Inhibitor Response 1; self-excising cassette.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism
  • CRISPR-Cas Systems
  • Caenorhabditis elegans / genetics*
  • Caenorhabditis elegans Proteins / metabolism
  • F-Box Proteins / chemistry
  • F-Box Proteins / genetics*
  • F-Box Proteins / metabolism
  • Genes, Reporter
  • Genetic Engineering / methods*
  • Indoleacetic Acids / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Organ Specificity
  • Proteolysis*
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / genetics*
  • Receptors, Cell Surface / metabolism
  • Transgenes

Substances

  • Arabidopsis Proteins
  • Caenorhabditis elegans Proteins
  • F-Box Proteins
  • Indoleacetic Acids
  • Luminescent Proteins
  • Receptors, Cell Surface
  • TIR1 protein, Arabidopsis