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bioRxiv · 10.64898/2026.05.06.723292

Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning

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

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.

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Isom, D. G., Pujols, J., Lee, K. D., Kapolka, N. J., Taylor, S., Colon, B.. 2026-05-07. Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning. https://doi.org/10.64898/2026.05.06.723292

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