{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,11,2]],"date-time":"2026-11-02T22:14:29Z","timestamp":1793657669391,"version":"4.5.1"},"posted":{"date-parts":[[2026,4,9]]},"group-title":"Genetics","reference-count":94,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T00:00:00Z","timestamp":1775692800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"name":"NIH","award":["K08 CA286740"],"award-info":[{"award-number":["K08 CA286740"]}]},{"name":"NIH","award":["DP2 CA311217"],"award-info":[{"award-number":["DP2 CA311217"]}]},{"id":[{"id":"https:\/\/ror.org\/01d35cw23","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/0184qbg02","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,9]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Genetic\n                  <jats:underline>engineering<\/jats:underline>\n                  experiments and therapies are constrained by the size of DNA integrations into human cell\u2019s genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond \u223c5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating &gt;5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA \u201chelper\u201d plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large\u2014up to 10 kb DNA\u2014integrations at &gt;20% efficiency in primary human T cells at the\n                  <jats:italic>TRAC<\/jats:italic>\n                  locus and at &gt;60% efficiency in human iPSCs at the\n                  <jats:italic>AAVS1<\/jats:italic>\n                  locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional\n                  <jats:italic>in vitro<\/jats:italic>\n                  and\n                  <jats:italic>in vivo<\/jats:italic>\n                  . Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.\n                <\/jats:p>","DOI":"10.64898\/2026.04.09.717505","type":"posted-content","created":{"date-parts":[[2026,4,9]],"date-time":"2026-04-09T23:05:16Z","timestamp":1775775916000},"source":"Crossref","is-referenced-by-count":3,"title":["Ultra-large targeted DNA integrations in primary human cells"],"prefix":"10.64898","author":[{"given":"Courtney","family":"Kernick","sequence":"first","affiliation":[{"name":"Department of Pathology, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Chow","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA"},{"name":"Gladstone-UCSF Institute 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