Accelerating research through tool development
We are always trying to drive our research forward, and when current tools are lacking we make new ones. We developed pha-1 co-conversion to very efficiently knock-in epitopes into endogenous loci using oligonucleotides and CRISPR/Cas9 (Ward, 2015). By selecting for repair of a temperature- sensitive, conditional lethal mutation, we were able to significantly enrich for knock-ins at other loci. Since the temperature sensitive allele is edited back to a wild-type allele, no selective markers remain in the genome. The stringency of this co-selection allowed optimization of numerous oligo-editing parameters (ie. homology length, DSB position, oligo polarity). This approach greatly facilitates editing of the C. elegans genome and is likely applicable to a range of model systems. We also previously collaborated with Dan Dickinson and Bob Goldstein to develop a methodology using homologous recombination and CRISPR/Cas9-induced DNA breaks to knock-in sequences into the endogenous C. elegans genes (Dickinson et al., 2013). Working with Abby Dernburg’s group (UC Berkeley) we have developed a conditional protein depletion system for use in C. elegans (Zhang et al., 2015). Introduction of a small, 44-amino acid tag (AID tag) onto a protein confers rapid degradation (20-60 minutes) of the tagged protein in animals both treated with a plant hormone, auxin, and expressing a plant E3 ligase (TIR1). TIR1 can be expressed ubiquitously or in a tissue-specific manner, allowing great flexibility in when and where a tagged protein is degraded. We are always looking to make these methods more efficient, cost-effective, and widely available.
The AID system enables functional analysis of nuclear hormone receptors during development. From Zhang et al., 2015. (A) Representative images of animals from the timed egg lay on control or 1 mM auxin plates following 60 h at 25°C. In the absence of auxin, no defects were seen in any genotype; however, in the presence of auxin, worms expressing degron-tagged NHR-25 and pan-somatic TIR1 showed molting defects (arrow indicated unshed cuticle) and gonadal defects such as tumorous germ lines (note lack of eggs and abnormal germ line). Animals expressing degron-tagged NHR-23 and pan-somatic TIR1 uniformly arrested as L1 larvae. Scale bar: 50 μm. (B) Temporal analysis of inducible protein degradation. Worms of the indicated genotypes were grown for six hours at 25°C following dauer release before 0.25% ethanol (control) or 1 mM auxin were added and samples harvested every 20 min. Lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Stain-free (Bio-Rad) analysis of total protein on each blot is provided as a loading control. Two isoforms of NHR-25 (a and b) are detected, as previously described (Ward, 2015).
