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Here, we develop an all-RNA platform for efficient, durable and multiplexed epigenetic programming in primary human T cells, stably turning endogenous genes off or on using CRISPRoff and CRISPRon epigenetic editors. We achieve epigenetic programming of diverse targeted genomic elements without the need for sustained expression of CRISPR systems. CRISPRoff-mediated gene silencing is maintained through numerous cell divisions, T cell stimulations and in vivo adoptive transfer, avoiding cytotoxicity or chromosomal abnormalities inherent to multiplexed Cas9-mediated genome editing. Lastly, we successfully combined genetic and epigenetic engineering using orthogonal CRISPR Cas12a\u2013dCas9 systems for targeted chimeric antigen receptor (CAR) knock-in and CRISPRoff silencing of therapeutically relevant genes to improve preclinical CAR-T cell-mediated in vivo tumor control and survival.<\/jats:p>","DOI":"10.1038\/s41587-025-02856-w","type":"journal-article","created":{"date-parts":[[2025,10,21]],"date-time":"2025-10-21T09:04:28Z","timestamp":1761037468000},"page":"1470-1481","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":43,"title":["Integrated epigenetic and genetic programming of primary human T cells"],"prefix":"10.1038","volume":"44","author":[{"given":"Laine","family":"Goudy","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7092-9072","authenticated-orcid":false,"given":"Alvin","family":"Ha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashir A.","family":"Borah","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer M.","family":"Umhoefer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lauren","family":"Chow","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carinna","family":"Tran","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aidan","family":"Winters","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6734-413X","authenticated-orcid":false,"given":"Alexis","family":"Talbot","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosmely","family":"Hernandez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhongmei","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2503-7200","authenticated-orcid":false,"given":"Sanjana","family":"Subramanya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0024-2937","authenticated-orcid":false,"given":"Abolfazl","family":"Arab","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3185-8646","authenticated-orcid":false,"given":"Nupura","family":"Kale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2998-5249","authenticated-orcid":false,"given":"Jae Hyun J.","family":"Lee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3784-3565","authenticated-orcid":false,"given":"Joseph J.","family":"Muldoon","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chang","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ralf","family":"Schmidt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7352-0339","authenticated-orcid":false,"given":"Philip","family":"Santangelo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9410-7148","authenticated-orcid":false,"given":"Julia","family":"Carnevale","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8262-1190","authenticated-orcid":false,"given":"Justin","family":"Eyquem","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9569-3708","authenticated-orcid":false,"given":"Brian R.","family":"Shy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2734-5776","authenticated-orcid":false,"given":"Alex","family":"Marson","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5854-0825","authenticated-orcid":false,"given":"Luke A.","family":"Gilbert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,10,21]]},"reference":[{"key":"2856_CR1","doi-asserted-by":"publisher","first-page":"1361","DOI":"10.1126\/science.aar6711","volume":"359","author":"CH June","year":"2018","unstructured":"June, C. 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A.M. is a cofounder of Site Tx, Arsenal Biosciences, Spotlight Therapeutics and Survey Genomics, serves on the boards of directors at Site Tx, Spotlight Therapeutics and Survey Genomics, is a member of the scientific advisory boards of network.bio, Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, Survey Genomics, NewLimit, Amgen and Tenaya, owns stock in network.bio, Arsenal Biosciences, Site Tx, Cellanome, Spotlight Therapeutics, NewLimit, Survey Genomics, Tenaya and Lightcast and has received fees from network.bio, Site Tx, Arsenal Biosciences, Cellanome, Spotlight Therapeutics, NewLimit, Abbvie, Gilead, Pfizer, 23andMe, PACT Pharma, Juno Therapeutics, Tenaya, Lightcast, Trizell, Vertex, Merck, Amgen, Genentech, GLG, ClearView Healthcare, AlphaSights, Rupert Case Management, Bernstein and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The A.M. laboratory has received research support from PICI, the Emerson Collective, Arc Institute, Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem and reagents from Genscript, Illumina, 10X Genomics, Ultima, and Cellanome. B.R.S is a compensated member of the scientific advisory board for Kano Therapeutics. B.R.S. is an inventor on patent applications pertaining to and based on the findings described in this paper, a subset of which have been licensed by the University of California. J.C. is a consultant for Waypoint Bio. J.E. is a compensated cofounder at Mnemo Therapeutics. J.E. owns stocks in Mnemo Therapeutics and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital, Resolution Therapeutics, Enterome and Treefrog Therapeutics. The J.E. lab has received research support from Cytovia, Therapeutics, Mnemo Therapeutics and Takeda Pharmaceutical Company. J.M.U. is a current employee of Site Tx. The remaining authors declare no competing interests.","order":1,"name":"Ethics","label":"Competing interests","group":{"name":"EthicsHeading","label":"Ethics"}}]}}