{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T09:12:34Z","timestamp":1778490754169,"version":"3.51.4"},"posted":{"date-parts":[[2026,5,7]]},"group-title":"Synthetic Biology","reference-count":46,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,7]],"date-time":"2026-05-07T00:00:00Z","timestamp":1778112000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"crossref","award":["R35GM119518"],"award-info":[{"award-number":["R35GM119518"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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\u2013coupled 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\n                  <jats:italic>in vivo<\/jats:italic>\n                  , and enabling scalable ground-truth benchmarking of predictive chimera engineering.\n                <\/jats:p>","DOI":"10.64898\/2026.05.06.723292","type":"posted-content","created":{"date-parts":[[2026,5,8]],"date-time":"2026-05-08T03:55:20Z","timestamp":1778212520000},"source":"Crossref","is-referenced-by-count":0,"title":["Systematic mapping of chimera-permissive sites by CRISPR-guided PAM scanning"],"prefix":"10.64898","author":[{"given":"Jacinda","family":"Pujols","sequence":"first","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"Molecular and Cellular Pharmacology Graduate Program"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kyutae D.","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"University of Miami Medical Scientist Training Program, University of Miami Miller School of Medicine, Miami, FL 33136, USA"},{"name":"Molecular and Cellular Pharmacology Graduate Program"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nick J.","family":"Kapolka","sequence":"additional","affiliation":[{"name":"EvE Bio, LLC, Durham, NC"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sam","family":"Taylor","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"The Sheila and David Fuente Graduate Program in Cancer Biology"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bruno","family":"Colon","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"Molecular and Cellular Pharmacology Graduate Program"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5637-2370","authenticated-orcid":false,"given":"Daniel G.","family":"Isom","sequence":"additional","affiliation":[{"name":"Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, Miami, FL, 33136, USA"},{"name":"Frost Institute for Data Science and Computing, Coral Gables, FL, 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