GSE185343
Genome-scale CRISPR screen reveals inhibitory factors as regulatory module of HIV
To identify host factors that restrict HIV replication, we conducted a CRISPR activation screen in a susceptible T cell line using a high-complexity, genome-wide sgRNA library. Our results identified host factors that conferred protection from HIV infection. Overall design: To identify cellular factors restricting HIV-1 replication, we utilized a previously engineered synergistic activation mediator complex (SAM) for the transcriptional activation of endogenous genes. dCas9 and MS2 expressing CD4 T cells were expanded and 300 million cells were further transduced with the SAM lentiviral library (3 sgRNAs per gene for 70,290 guides in total) at an MOI = 0.3 for 7 days. The gain-of-expression cells were divided into two pools. We challenged the first pool of 60 million transduced cell with HIV at an MOI of 1 for 4 days and then treated cells with GCV for 3 days to further deplete infected cells. Two rounds of infection and GCV selection were performed to enrich HIV resistant cells to identify host genes important in HIV infection. The other pool of 30 million cells were mock infected as control. After 2 weeks, surviving cells were sorted out. Genomic DNA was extracted from harvested cells and sgRNAs were PCR-amplified and deep sequenced. Normalized reads of HIV infected cells were compared to mock infected cells.