Efficient generation of a single-copy eft-3p::TIR1::F2A:: BFP::AID*::NLS allele in the C. elegans ttTi5605 insertion site through recombination-mediated cassette exchange

Abstract

The auxin-inducible degron (AID) system is a widely used system to conditionally deplete proteins. Using CRISPR/Cas9-based genome editing in C. elegans, we recently generated a set of single-copy, tissue-specific and pan-somatic TIR1-expressing strains carrying a BFP reporter inserted in single-copy into two commonly used, well-characterized genetic loci. However, we were unable to obtain a strain carrying a pan-somatic eft-3p::TIR1::F2A::BFP::AID*::NLS transgene inserted into the chromosome II ttTi5605 insertion site. Using recombination-mediated cassette exchange (RMCE) we were able to efficiently obtain this knock-in. The resulting strain displayed equivalent depletion of an AID*::GFP reporter compared to our previously generated eft-3p::TIR1::F2A::BFP::AID*::NLS transgene knocked into the chromosome I ttTi4348 insertion site. This work highlights the power of RMCE for generating new reagents for the AID system and provides an eft-3p::TIR1::F2A::BFP::AID*::NLS allele on chromosome II which will simplify genetic crossing schemes when using the AID system.

Figure 1. Validation of a chromosome II single-copy eft-3p::TIR1::F2A::BFP::AID*::NLS transgene

eft-3p::TIR1::F2A::BFP::AID*::NLS animals (late L3 stage) were shifted onto control or 4 mM K-NAA (1-Naphthaleneacetic Acid, Potassium Salt) auxin plates for the indicated times (0, 30, 60, 120 minutes). (A) Animals were collected and DIC, BFP, and GFP imaging of the head region was performed. Scale bars represent 10 µm. (B) Western blots detecting AID*::GFP in control- or auxin (4 mM K-NAA)-treated animals. Anti-GFP blots (top) detected background bands (marked with *) also reported by Ashley et al., 2021 and a doublet at approximately 27 kDa, consistent with the predicted size of GFP. Total protein is provided as a loading control. Equal exposure times were used to collect each set of GFP and BFP images.