Degron models: a toolbox for rapid in vivo depletion of essential proteins regulating mRNA metabolism

Due to their essentiality, studying proteins involved in fundamental processes in vivo is challenging. PROTAC-based systems offer time-controlled protein depletion, but their characterization in vivo remains limited. Here, with an efficient direct zygote editing protocol, we generated degron-tag models (dTAG/FKBP or BromoTag) for seven genes involved in therapeutic and endogenous mRNA metabolism (Cnot1, Pan2, Tent5a, Tent4b, Dcp2, Rnasel, Tsg101). Degron tags occasionally caused phenotypes that were often mitigated by tag position or tagging system change. In cells, both approaches yielded rapid and sustained degradation. In mice, dTAG depletion was effective but varied by protein and administration route, whereas BromoTag showed no in vivo activity. We showcase the utility of these models through an analysis of CNOT1's roles in cell division, immunity, and poly(A) tail maintenance. We present a valuable toolbox for studying mRNA metabolism in mammalian models, while providing a benchmark for applying degron-tag models to study other biological processes. Overall design: This study includes mouse liver and primary cell (BMDM) samples derived from genetically engineered mouse lines expressing FKBP-tagged proteins (CNOT1, TENT5A, TENT4B, RNASEL, PAN2, DCP2). Experimental conditions include treatment with small-molecule degrader (dTAGV-1) versus vehicle controls (DMSO). Samples were collected at multiple time points after treatment. Control samples include wild-type littermates and vehicle-treated tagged mice or cells. Biological replicates were included for each experimental condition.