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

Protein chimeras enable new biological functions by combining otherwise independent protein elements, but identifying insertion sites that preserve host protein function remains largely empirical. Here, we introduce CRISPR-guided protospacer adjacent motif (PAM) scanning, a scalable strategy for mapping chimera-permissive sites directly in living cells. PAM scanning uses CRISPR-addressable positions across coding sequences to systematically install functional payloads and measure insertion tolerance. In yeast, PAM accessibility provided near-comprehensive coverage across proteins of diverse size and function. We first applied this approach across an entire G protein-coupled receptor (GPCR) signaling pathway, generating 91 chimeras containing inserted protease recognition sequences to map insertion tolerance and site-specific biophysical protection from a protease probe by surrounding protein structure and binding partners. Cognate protease cleavage revealed how this protection varied with protease inhibition and size. We then used PAM scanning to identify bi-functional GPCR chimeras that retained receptor signaling while supporting fluorescent or luminescent reporters. Screening human adenosine A2A and melatonin MTNR1A receptors in yeast identified functional mNeonGreen and NanoLuc insertion sites that retained ligand-dependent signaling following translation to human cells. Together, these studies establish PAM scanning as a general framework for mapping insertional tolerance and biophysical protection in living cells, accelerating functional chimera engineering, and experimentally validating generative chimera design.