{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:27:37Z","timestamp":1786508857624,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,10]]},"group-title":"Genomics","reference-count":91,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["RM1-HG011123"],"award-info":[{"award-number":["RM1-HG011123"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01-MH125236"],"award-info":[{"award-number":["R01-MH125236"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UM1-HG012053"],"award-info":[{"award-number":["UM1-HG012053"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>CRISPR technologies based on nuclease-deactivated Cas9 (dCas9) rely on programmable DNA binding rather than DNA cleavage, yet the intrinsic DNA-recognition properties that govern optimal guide RNA (gRNA) performance remain poorly understood. Existing approaches either measure genomic occupancy in cells or infer dCas9 behavior from cleavage-based Cas9 datasets, despite DNA binding being substantially more permissive than DNA cleavage. Here we introduce TANGO (Targeted Array-based Nucleic acid-Guided Occupancy), a high-density DNA-array platform that quantitatively profiles intrinsic dCas9:gRNA binding across tens of thousands of DNA targets in a cell-free system. TANGO captures established features of dCas9 target recognition, while providing substantially greater sensitivity than prior assays. Comparison with ChIP-seq data demonstrates that intrinsic DNA-binding specificity is a major driver of genomic occupancy and reveals that chromatin accessibility modulates the intrinsic binding affinity required for dCas9 recruitment. Across CRISPRi\/a guides, TANGO identifies multiple independent biochemical determinants of guide performance\u2014including on-target affinity, mismatch tolerance, and ribonucleoprotein assembly\u2014and flags problematic and highly promiscuous guides overlooked by current specificity metrics. Unexpectedly, some guides retain substantial guide-directed DNA binding even in the absence of a protospacer-adjacent motif (PAM), revealing an additional dimension of dCas9 specificity. Together, these results establish intrinsic DNA recognition as a quantitative and experimentally accessible determinant of dCas9 function, providing a framework for improving guide selection and enhancing the precision of CRISPR technologies.<\/jats:p>","DOI":"10.64898\/2026.08.10.743836","type":"posted-content","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T05:05:17Z","timestamp":1786424717000},"source":"Crossref","is-referenced-by-count":0,"title":["Quantitative profiling of intrinsic dCas9-DNA recognition reveals key determinants of guide RNA performance"],"prefix":"10.64898","author":[{"given":"Wei","family":"Zhu","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Duke University, Durham, NC, USA"},{"name":"Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA"},{"name":"Program in Cellular and Molecular Medicine, Boston Children\u2019s Hospital, Boston, MA, 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USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jameson","family":"Blount","sequence":"additional","affiliation":[{"name":"Program in Computational Biology and Bioinformatics, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexias","family":"Safi","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Duke University, Durham, NC, USA"},{"name":"Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jacob","family":"Schreiber","sequence":"additional","affiliation":[{"name":"Department of Genomics and Computational Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charles A.","family":"Gersbach","sequence":"additional","affiliation":[{"name":"Center for Advanced Genomic Technologies, Duke University, Durham, NC, 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