{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:45:27Z","timestamp":1768509927820,"version":"3.49.0"},"posted":{"date-parts":[[2023,12,14]]},"group-title":"Immunology","reference-count":46,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,12,14]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges\n                  <jats:sup>1<\/jats:sup>\n                  . Here, we developed a high-throughput approach for high-efficiency and massively parallel adenine and cytosine base-editor screening in primary human T cells. We performed multiple large-scale screens editing 102 genes with central functions in T cells and full-length tiling mutagenesis of selected genes, and read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. We discovered a broad landscape of gain- and loss-of-function mutations, including in\n                  <jats:italic>PIK3CD<\/jats:italic>\n                  and its regulatory subunit encoded by\n                  <jats:italic>PIK3R1, LCK<\/jats:italic>\n                  ,\n                  <jats:italic>AKT1, CTLA-4<\/jats:italic>\n                  and\n                  <jats:italic>JAK1<\/jats:italic>\n                  . We identified variants that affected several (e.g.,\n                  <jats:italic>PIK3CD<\/jats:italic>\n                  C416R) or only selected (e.g.\n                  <jats:italic>LCK<\/jats:italic>\n                  Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation. Using primary human T cells in which we engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, we demonstrate that base edits identified in our screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. In summary, we present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at high efficiency. Coupled with multi-modal phenotypic mapping, we accurately nominate variants that produce a desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies.\n                <\/jats:p>","DOI":"10.1101\/2023.12.13.571465","type":"posted-content","created":{"date-parts":[[2023,12,14]],"date-time":"2023-12-14T13:20:43Z","timestamp":1702560043000},"source":"Crossref","is-referenced-by-count":0,"title":["Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells"],"prefix":"10.64898","author":[{"given":"Zachary H.","family":"Walsh","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Parin","family":"Shah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neeharika","family":"Kothapalli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gergo","family":"Nikolenyi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shivem B.","family":"Shah","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Giuseppe","family":"Leuzzi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael","family":"Mu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patricia","family":"Ho","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sinan","family":"Abuzaid","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zack D.","family":"Brodtman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Neil","family":"Vasan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammed","family":"AlQuraishi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joshua D.","family":"Milner","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Alberto","family":"Ciccia","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Johannes C.","family":"Melms","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2379-6702","authenticated-orcid":false,"given":"Benjamin","family":"Izar","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023121718500800000_2023.12.13.571465v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-022-01765-8"},{"key":"2023121718500800000_2023.12.13.571465v1.2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaa4967"},{"key":"2023121718500800000_2023.12.13.571465v1.3","doi-asserted-by":"crossref","unstructured":"Ruella M , Korell F , Porazzi P , Maus MV . 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