{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T11:50:08Z","timestamp":1768477808209,"version":"3.49.0"},"posted":{"date-parts":[[2017,10,19]]},"group-title":"Systems Biology","reference-count":66,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2017,10,19]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  CRISPR-Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human T cells to generate a high-throughput platform for analyzing the role of host factors in pathogen infection and lifecycle. Here, we describe applications of this system to investigate HIV pathogenesis in CD4+ T cells. Briefly, CRISPR-Cas9 ribonucleoproteins (crRNPs) are synthesized\n                  <jats:italic>in vitro<\/jats:italic>\n                  and delivered to activated primary human CD4+ T cells by nucleofection. These edited cells are then validated and expanded for use in downstream cellular, genetic, or protein-based assays. Our platform supports the arrayed generation of several gene manipulations in only a few hours\u2019 time and is widely adaptable across culture conditions, infection protocols, and downstream applications. We present detailed protocols for crRNP synthesis, primary T cell culture, 96-well nucleofection, molecular validation, and HIV infection with additional considerations for guide and screen design as well as crRNP multiplexing.\n                <\/jats:p>","DOI":"10.1101\/205500","type":"posted-content","created":{"date-parts":[[2017,10,20]],"date-time":"2017-10-20T01:10:32Z","timestamp":1508461832000},"source":"Crossref","is-referenced-by-count":1,"title":["A CRISPR-Cas9 Genome Engineering Platform in Primary CD4+ T Cells for the Interrogation of HIV Host Factors"],"prefix":"10.64898","author":[{"given":"Judd F.","family":"Hultquist","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8015-9614","authenticated-orcid":false,"given":"Joseph","family":"Hiatt","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kathrin","family":"Schumann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Michael J.","family":"McGregor","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Theodore 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