Phenotypic assessment of protein function and target validation using reversible knock-out of endogenously expressed protein targets via a fusion tag-directed PROTAC
This talk will describe a target validation approach based on reversible and conditional live-cell knockout of endogenously expressed proteins at the protein level, as opposed to genomic level. We will highlight the use of CRISPR/CAS9 techniques to insert HaloTag fusions into endogenous genomic loci alone or in tandem with the 11 amino-acid HiBiT tag to achieve sensitive luminescent detection of protein expression without the use of antibodies. Through use of a HaloTag-directed PROTAC (HaloPROTAC3) to trigger target protein degradation via recruitment of the E3 ligase component VHL and the ubiquitin-proteasome degradation pathway, we will demonstrate rapid functional knockout of endogenous protein targets in various subcellular compartments.
