{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T13:11:15Z","timestamp":1785417075831,"version":"3.56.0"},"posted":{"date-parts":[[2025,10,29]]},"group-title":"Genomics","reference-count":70,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T00:00:00Z","timestamp":1761696000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R01 GM137606"],"award-info":[{"award-number":["R01 GM137606"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35 GM148220"],"award-info":[{"award-number":["R35 GM148220"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35 GM122569"],"award-info":[{"award-number":["R35 GM122569"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["U01 DK127432"],"award-info":[{"award-number":["U01 DK127432"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["S10 OD026839"],"award-info":[{"award-number":["S10 OD026839"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F31 GM148042"],"award-info":[{"award-number":["F31 GM148042"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,10,29]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6\u00d7NLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6\u00d7NLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9\u00b7gRNA complex to study its inhibitory mechanism. 6\u00d7NLS-Acr (IC\n                  <jats:sub>50<\/jats:sub>\n                  = 0.47 \u00b5M) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6\u00d7NLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6\u00d7NLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.\n                <\/jats:p>","DOI":"10.1101\/2025.10.28.685205","type":"posted-content","created":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T22:20:19Z","timestamp":1761776419000},"source":"Crossref","is-referenced-by-count":0,"title":["Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4522-2793","authenticated-orcid":false,"given":"Axel O.","family":"Vera","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7367-6077","authenticated-orcid":false,"given":"Franklin J.","family":"Avil\u00e9s-V\u00e1zquez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2195-6258","authenticated-orcid":false,"given":"Taekjip","family":"Ha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8437-0150","authenticated-orcid":false,"given":"Amit","family":"Choudhary","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7164-1719","authenticated-orcid":false,"given":"Ronald T.","family":"Raines","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025103107500461000_2025.10.28.685205v1.1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1138140"},{"key":"2025103107500461000_2025.10.28.685205v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"2025103107500461000_2025.10.28.685205v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1231143"},{"key":"2025103107500461000_2025.10.28.685205v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"2025103107500461000_2025.10.28.685205v1.5","doi-asserted-by":"publisher","DOI":"10.1056\/nejmoa2031054"},{"key":"2025103107500461000_2025.10.28.685205v1.6","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2107454"},{"key":"2025103107500461000_2025.10.28.685205v1.7","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2300709"},{"key":"2025103107500461000_2025.10.28.685205v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2623"},{"key":"2025103107500461000_2025.10.28.685205v1.9","doi-asserted-by":"publisher","DOI":"10.1126\/science.aav9973"},{"key":"2025103107500461000_2025.10.28.685205v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-023-01859-2"},{"key":"2025103107500461000_2025.10.28.685205v1.11","doi-asserted-by":"crossref","first-page":"1143157","DOI":"10.3389\/fbioe.2023.1143157","article-title":"Off-target effects in CRISPR\/Cas9 gene editing","volume":"11","year":"2023","journal-title":"Front. 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