{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T20:54:13Z","timestamp":1768510453939,"version":"3.49.0"},"posted":{"date-parts":[[2024,2,14]]},"group-title":"Bioengineering","reference-count":46,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2024,2,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the\n                  <jats:italic>KLRC1<\/jats:italic>\n                  gene (encoding the HLA-E-binding NKG2A receptor) and perform non-viral insertion of a GD2-targeting chimeric antigen receptor (CAR) within NK cells isolated from human peripheral blood. Genome editing with CRISPR\/Cas9 ribonucleoprotein complexes yields efficient genomic disruption of the\n                  <jats:italic>KLRC1<\/jats:italic>\n                  gene with 98% knockout efficiency and specific knock-in of the GD2 CAR transgene as high as 23%, with minimal off-target activity as shown by CHANGE-Seq, in-out PCR, and next generation sequencing.\n                  <jats:italic>KLRC1<\/jats:italic>\n                  -GD2 CAR NK cells display high viability and proliferation, as well as precise cellular targeting and potency against GD2\n                  <jats:sup>+<\/jats:sup>\n                  human melanoma cells. Notably,\n                  <jats:italic>KLRC1<\/jats:italic>\n                  -GD2 CAR NK cells overcome HLA-E-based inhibition by HLA-E-expressing, GD2\n                  <jats:sup>+<\/jats:sup>\n                  melanoma cells. Using a single-step, virus-free genome editing workflow, this study demonstrates the feasibility of precisely disrupting inhibitory signaling within NK cells via CRISPR\/Cas9 while expressing a CAR to generate potent allogeneic cell therapies against HLA-E\n                  <jats:sup>+<\/jats:sup>\n                  solid tumors.\n                <\/jats:p>","DOI":"10.1101\/2024.02.14.580371","type":"posted-content","created":{"date-parts":[[2024,2,14]],"date-time":"2024-02-14T23:00:13Z","timestamp":1707951613000},"source":"Crossref","is-referenced-by-count":0,"title":["Virus-free CRISPR knock-in of a chimeric antigen receptor into\n                  <i>KLRC1<\/i>\n                  generates potent GD2-specific natural killer cells"],"prefix":"10.64898","author":[{"given":"Keerthana","family":"Shankar","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Isabella","family":"Zingler-Hoslet","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lei","family":"Shi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Varun","family":"Katta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brittany E.","family":"Russell","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shengdar Q.","family":"Tsai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2276-6731","authenticated-orcid":false,"given":"Christian M.","family":"Capitini","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2837-0858","authenticated-orcid":false,"given":"Krishanu","family":"Saha","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2024021707400584000_2024.02.14.580371v1.1","doi-asserted-by":"publisher","DOI":"10.1056\/nejmra1706169"},{"key":"2024021707400584000_2024.02.14.580371v1.2","doi-asserted-by":"crossref","first-page":"1002","DOI":"10.1056\/NEJMoa2213614","article-title":"Ide-cel or Standard Regimens in Relapsed and Refractory Multiple Myeloma","volume":"388","year":"2023","journal-title":"N. 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