Genome-Wide CRISPR Screen of T Cell Function

Nine shRNA pools (~5 shRNAs per gene) were created and subcloned into the pLKO-Thy1.1 lentiviral vector. Each pool also included 85 negative-control shRNAs. OT-I T cells were cultured with IL-7 (5ng/mL) and IL-15 (100ng/mL); on day 2 cells were spin-infected with lentiviral pools supplemented with protamine sulfate (5µg/mL) in retronectin-coated 24-well plates (5µg/mL) at a multiplicity of infection (MOI) of 15. Following infection, OT-1 cells were cultured with IL-7 (2.5ng/mL), IL-15 (50ng/mL) and IL-2 (2ng/mL). On day 5, shRNA-transduced T cells were enriched by positive selection using the Thy1.1 surface reporter (Stemcell Technologies). T cells (5×10⁶) were injected i.v. into C57BL/6 mice bearing day 14 B16-Ova tumors (15 mice per shRNA pool). Seven days later, shRNA-expressing T cells (CD8⁺Vα2⁺Vβ5⁺Thy1.1⁺) were isolated by flow cytometry from tumors, spleens, tumor-draining lymph nodes and irrelevant lymph nodes. Genomic DNA was purified (Qiagen) and deep-sequencing templates were generated by PCR amplification of the shRNA cassette. Representation of shRNAs in each pool was analyzed by deep sequencing using an Illumina Genome Analyzer^{47 (link)}.
Secondary screens were performed using focused pools containing ~15 shRNAs/gene as well as 85 negative controls. Cut-off in the secondary screen was defined as >3 shRNAs with >4 fold enrichment in tumor relative to spleen. Screening results were validated at a cellular level by introducing individual shRNAs into T cells, along with a reporter protein (GFP, TFP, RFP or Ametrine fluorescent proteins, Thy1.1). This approach enabled simultaneous testing of five shRNAs in an animal (three mice per group). Proliferation of shRNA-transduced T cells was visualized based on CFSE dilution after 24 hours as well as 3, 5 and 7 days.