CRISPR screen using the GeCKO library

We utilized a CRISPR-based approach to identify novel host proteins that are involved in regulating HIV gene expression and viral latency. To this end, we generated Jurkat cells that express Cas9-Flag (S6 Fig, showing western blot for Cas9). Stable cells were then transduced with a lentivirus expressing HIV-Tat-BFP and sorted on the basis of their reporter expression at 72 h post transduction to obtain a cell population that expressed BFP. Thereafter, 1.3×10⁸ cells were transduced with a lentivirus that expressed the GeCKO whole genome sgRNA library (number of cells corresponded to ×300 the size of the GeCKO library at MOI of 0.3 infection). GeCKO (a generous gift from the laboratory of Ophir Shalem) consists of multiple sgRNA guides that target a total of 19,052 human genes. For each gene, there are six sgRNA constructs, along with 1864 sgRNAs against miRNA with 4 constructs each, making a total library of 122,417 sgRNA members. Cell transduction of GeCKO lentivirus was performed at an MOI of 0.3, so as to statistically ensure the integration of a single sgRNA per cell and to achieve ×300 library coverage (library size 1.3×10⁵). Following transduction, cells were subjected to puromycin selection to obtain stable cells that expressed both Cas9 and the sgRNA GeCKO library. All cells expressed the BFP reporter gene and were thus transduced with HIV. As a control, cells were also transduced with a lentivirus that expressed scramble sgRNA. GeCKO-expressing cells or control cells were then grown over a period of 30–40 days, allowing them to enter latency, as detected by the loss of BFP expression. Thereafter, 40×10⁶ GeCKO-expressed cells (×300 of library size of 1.3×10⁵ members) or control cells were analyzed by FACS for BFP expression. In the GeCKO expressed cells, 44% were BFP(+), while 45% were BFP(–). In the control cells that expressed scramble sgRNA, 22% were BFP(+) and 76% were BFP(–). From each group of cells, 12×10⁶ (×100 above library size) cells were sent for NGS analysis. sgRNA enrichment was analyzed by the CRISPR analyzer and compared for each group of cells vs the control cells that expressed the entire GeCKO library.