Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule

Abstract

Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20–25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Targeted Protein Degradation by Small Molecules

“…This system makes used of a destabilized mutant of FKBP12 that is stabilized in the presence of the compound Shld-1 and has proved useful in controlling fusion protein stability in parasites (95), worms (96), and medaka (97) as well as to potassium channel biology (98) and the cytosolic UPR in mammalian cells (99). Several technological advancements are worth noting: By combining Shld-1 stabilization with induced dimerization of a split-ubiquitin system, release of the native protein is achieved in response to a small molecule (100).…”

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

“…1E). Fusing I-SceI to an ecDHFR degradation domain (ecDHFR-I-SceI) allows I-SceI protein stabilization after addition of the ligand trimethoprim (Cho et al 2013). Hsp70.I-SceI is a heat-shock-inducible system with hsp70 promoter-dependent I-SceI expression ( Fig.…”

Small-molecule control of super-Mendelian inheritance in gene drives

“…Upon expression, the DD-SpCas9 fusion protein is targeted for proteasomal degradation unless the DHFR-binding small molecule, trimethoprim (TMP), is added. Because destabilized domains are efficacious in multiple organisms [15][16][17][18] , regulated by a synthetic molecule, provide superior control of DNA-binding proteins over transcription-based controllers 19 , and can control SpCas9 in mammalian cells 14 , we reasoned that this system would provide an ideal gene-drive controller. Furthermore, as the small molecule is required to stabilize DD-SpCas9 in this default-off system, there is reversible dosage control of SpCas9 nuclease activity and an added level of safety in case of escaped organisms ( Fig.…”