Isom, D. G.; Pujols, J.; Lee, K. D.; Kapolka, N. J.; Taylor, S.; Colon, B.

Engineered protein chimeras enable new biological functions but remain difficult to design due to context-dependent constraints on insertion tolerance and the need to preserve host protein function. Here, we report CRISPR-guided protospacer adjacent motif (PAM) scanning in yeast to map chimera-permissive sites in living cells. We apply this approach to peptide and reporter insertions. In the first application, we generated 91 insertion chimeras encoding a defined protease cleavage sequence across six components of a model G protein-coupled receptor (GPCR) signaling pathway. Sixty-three percent of sites retain signaling, identifying positions that preserve host function and reveal broad, position-dependent tolerance. Coupling insertional scanning with cognate proteases enables site-resolved mapping of in-cell accessibility, distinguishing protected and exposed regions and defining EX1- and EX2-like regimes. These chimeras are responsive to proteolysis and pharmacological inhibition, enabling reversible control of protein activity. In a second application, we scanned 32 positions in yeast Ste2 and human A2A and MTNR1A receptors to engineer bi-functional chimeras that retain native function while incorporating reporter activity. Together, these results establish PAM scanning as a scalable, protein-agnostic framework for mapping insertion tolerance, interrogating protein accessibility in vivo, and enabling scalable ground-truth benchmarking of predictive chimera engineering.