Description

The AID system has emerged as a powerful tool to conditionally deplete proteins in a wide-range of organisms and cell types (Nishimura et al. 2009; Holland et al. 2012; Zhang et al. 2015; Natsume et al. 2016; Trost et al. 2016; Brown et al. 2017; Daniel et al. 2018; Chen et al. 2018; Camlin and Evans 2019). The system is comprised of two components. A plant F-box protein Transport Inhibitor Response 1 (TIR1) is expressed and forms a complex with endogenous Skp1 and Cul1 proteins to form a functional SCF ubiquitin ligase (Nishimura et al. 2009; Natsume and Kanemaki 2017). TIR1 can either be expressed constitutively or in a tissue-specific manner depending on promoter choice. A degron sequence from the IAA17 protein is fused to the protein of interest (Nishimura et al. 2009; Natsume and Kanemaki 2017). Commonly used auxin-inducible degrons include 44 amino acid (AID*) and 68 amino acid (mAID) fragments of IAA17 (Morawska and Ulrich 2013; Li et al. 2019). Addition of the plant hormone auxin bridges an interaction between TIR1 and the degron and the SCF ligase ubiquitylates the degron-fused protein leading to proteasomal degradation.

We recently described a new set of strains and reagents for the C. elegans AID system (Ashley et al. 2021), complementing the original version of the system, which used an mRuby2 fusion to monitor the expression of TIR1 (Zhang et al. 2015). We developed a TIR1::F2A::BFP::AID*::NLS transgene (Ashley et al. 2021) in which an F2A ribosome skip sequence allows production of separate TIR1 and BFP::AID*::NLS proteins from a single mRNA transcript (Ryan and Drew 1994; Ryan et al. 1999; Donnelly et al. 2001; de Felipe et al. 2003; Ahier and Jarriault 2014). The use of a BFP reporter allows for depletion experiments while imaging red and green fluorescent proteins (Ragle et al. 2020). TIR1 expression can be monitored by nuclear-localized BFP and the AID* sequence allows detection of TIR1 activity by auxin-dependent BFP depletion (Ashley et al. 2021). We previously inserted tissue-specific (germline, intestine, neuron, muscle, hypodermis, seam cell) TIR1::F2A::BFP::AID*::NLS transgenes into standardized genetic loci on chromosomes I and II (ttTi4348 and ttTi5605 Mos insertion sites, respectively) using CRISPR/Cas9-mediated genome editing (Frøkjaer-Jensen et al. 2008; Frøkjær-Jensen et al. 2012; Ashley et al. 2021). We generated a pan-somatic eft-3p:: TIR1::F2A::BFP::AID*::NLS transgene in the chromosome I site (Ashley et al. 2021), but were unable to generate an equivalent insertion in chromosome II despite injecting over two hundred animals. All other insertions were obtained by injecting 60 animals or fewer. As the pan-somatic TIR1 strains are widely requested (Aric Daul, Caenorhabditis Genetics Center (CGC), personal communication) we attempted to generate a chromosome II pan-somatic TIR1 strain through recombination-mediated cassette exchange (RMCE), an alternate genome editing method (Nonet 2020).

RMCE uses a germline-expressed Flp recombinase to insert transgenes into specialized integration landing sites (Nonet 2020). As there are now a number of landing sites, a single integration vector can be used to generate insertions into multiple genomic loci. We generated an integration vector containing an eft-3p::TIR1::F2A::BFP::AID*::NLS transgene, injected it into a strain carrying a landing pad in the ttTi5605 insertion site in chromosome II (M. Nonet, https://sites.wustl.edu/nonetlab/rmce-insertion-strains), and recovered a single knock-in from 30 injected animals on our first attempt. We expect that we can improve on this knock-in rate and move closer to the reported efficiency of injecting seven animals to achieve a knock-in (Nonet, 2020). We then generated an eft-3p::TIR1::F2A::BFP::AID*::NLS; eft-3p::AID*::GFP strain and performed a depletion time course to validate the TIR1 allele. At the 0 minute timepoint, we observed nuclear-localized BFP, indicating that the eft-3p::TIR1::F2A::BFP::AID*::NLS transgene was being transcribed and translated (Figure 1A). In control treated animals we observed no detectable change in BFP or GFP by microscopy (Figure 1A). In contrast, animals exposed to 4 mM auxin displayed a severe reduction of BFP and GFP by 120 minutes post-treatment (Figure 1A). We confirmed these results with an anti-GFP western blot (Figure 1B). GFP levels were stable in control animals, but dropped after 30 minutes of auxin exposure and were almost undetectable after two hours of exposure, similar to what we reported for our chromosome I eft-3p:: TIR1::F2A::BFP::AID*::NLS transgene (Ashley et al. 2021). This new TIR1 strain should complement our original chromosome I eft-3p::TIR1::F2A::BFP::AID*::NLS strain and provide a new allele on a separate chromosome to facilitate genetic crossing schemes. This work also highlights the effectiveness of the RMCE method for generation of single-copy transgenes at defined loci (Nonet 2020).