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We developed a new latency CRISPR screening strategy, called Latency HIV-CRISPR which uses the packaging of guideRNA-encoding lentiviral vector genomes into the supernatant of budding virions as a direct readout of factors involved in the maintenance of HIV-1 latency. We developed a custom guideRNA library targeting epigenetic regulatory genes and paired the screen with and without a latency reversal agent\u2013AZD5582, an activator of the non-canonical NF\u03baB pathway\u2013to examine a combination of mechanisms controlling HIV-1 latency. A component of the Nucleosome Acetyltransferase of H4 histone acetylation (NuA4 HAT) complex, ING3, acts in concert with AZD5582 to activate proviruses in J-Lat cell lines and in a primary CD4+ T cell model of HIV-1 latency. We found that the knockout of\n                    <jats:italic>ING3<\/jats:italic>\n                    reduces acetylation of the H4 histone tail and BRD4 occupancy on the HIV-1 LTR. However, the combination of\n                    <jats:italic>ING3<\/jats:italic>\n                    knockout accompanied with the activation of the non-canonical NF\u03baB pathway via AZD5582 resulted in a dramatic increase in initiation and elongation of RNA Polymerase II on the HIV-1 provirus in a manner that is nearly unique among all cellular promoters.\n                  <\/jats:p>","DOI":"10.1371\/journal.ppat.1011101","type":"journal-article","created":{"date-parts":[[2023,1,27]],"date-time":"2023-01-27T14:02:10Z","timestamp":1674828130000},"page":"e1011101","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":34,"title":["A modular CRISPR screen identifies individual and combination pathways contributing to HIV-1 latency"],"prefix":"10.1371","volume":"19","author":[{"given":"Emily","family":"Hsieh","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Derek H.","family":"Janssens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patrick J.","family":"Paddison","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Edward 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