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Institutehttps://www.ebi.ac.uk/ena/browser/view/PRJNA1080041Small molecule-triggered protein degradation tags (degrons) are powerful tools for biology, biotechnology, and therapeutics development. Commonly used degrons, however, are large protein domains (typically >100 amino acids) that cannot be easily or cleanly installed in endogenous protein-coding genes in most cell types. Although some zinc-finger (ZF) degrons are theoretically small enough for facile endogenous tagging, all are triggered by small molecules with substantial off-target effects, precluding their use as highly specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glues (MG-PACE) and applied it to evolve ZF degrons that engage cereblon (CRBN) only in the presence of orthogonal thalidomide derivatives. MG-PACE evolved a 36-amino acid ZF degron (SD40) that binds CRBN with high affinity in the presence of PT-179, a thalidomide derivative with no detected neosubstrate activity. The evolved degron is small enough to be efficiently inserted into targeted genomic sites in human cells using prime editing. Human proteins tagged with the evolved degron are rapidly and potently degraded by the otherwise inert PT-179. To overcome the inactivity of IMiD-based degrons in rodents, we separately used MG-PACE to evolve ZF degrons that engage mouse CRBN, enabling induced target protein degradation in mouse cells. High-resolution cryo-electron microscopy structures of SD40 in complex with CRBN-DDB1 bound to PT-179 or pomalidomide reveal mechanistic insights into the evolution and molecular basis of SD40's activity and specificity. Collectively, our efforts establish a system for the rapid evolution of molecular glue complexes with novel specificities and compact ZF tags that overcome shortcomings associated with existing degrons.ENAfalseContinuous evolution of compact protein degradation tags regulated by selective cereblon molecular glues2024-06-232024-06-23PRJNA1080041