Targeted protein degradation using conditional degron tags (CDTs) is an attractive strategy for post-translational manipulation of protein levels to interrogate biological function and therapeutic potential of proteins of interest, in cells and in vivo. Many CDT technologies have been developed, but finding the right tag for a protein of interest can often be an ad hoc and iterative process. To make matters worse, a systematic evaluation of available CDTs is not available to guide which one might be better suited for a particular target. Daniel Bondeson, a postdoctoral researcher at the Broad Institute of MIT and Harvard, and colleagues sought to address these concerns.
“No head-to-head comparison, or even the ability to make such a comparison, was available, and we thought there were two possibilities. First, it could be that one tag prevailed over the others and should be the strategy moving forward, or, second, that each tag would work with a few proteins but not all, and so systematically testing several strategies would always be the most effective path,” says Bondeson. They developed a systematic panel of lentiviral vectors to enable comparison of the tag technologies, as both N- and C-terminal fusions and with both strong and weak promoters. They tested the five most widely used technologies (AID, dTAG, IKZF3 degron, HaloTag and SMASh) on 16 different proteins spanning different protein classes, sizes and localizations. None of the CDTs were efficient across all targets, but the assessment allowed them to determine at least one optimal CDT fusion for each.
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