CRISPR screen identifies loss of IFNγR signaling and downstream adhesion as a resistance mechanism to CAR T-cell cytotoxicity in solid tumors

ABSTRACT: U87-CBG-GFP cells selected with blasticidin to stably express Cas9 were transduced with an MOI (< 1) of Brunello guide library (with puromycin resistance in the guide backbone). Cells were rested for 2 days then selected with puromycin antibiotic until the control, untransduced arm was killed (6-7 days). The guide-transduced cells were then pooled and replated in preparation for the screen. There were 12 arms to the screen, two control arms performed in technical duplicate and 8 sample arms from 6 normal donor CAR T-cells (2 were run in technical duplicate). At the time of CAR T-cell addition 2 arms were taken down as technical duplicates to measure guide frequency at the beginning of the screen. 2 additional arms received no CAR T-cells to serve as proliferation controls and were passaged the same as the sample CAR T conditions about to be described. CAR T-cells were added at a 1:10 effector:target cell ratio. After 18 hours of co-culture the CAR T-cells were removed by aspiration and the tumor cells were gently rinsed 4 times with PBS to remove any remaining CAR T-cells. The tumor cells were then passaged so that any remaining dying cells would not survive. After a 2-day recovery period, the tumor cells were rinsed once with PBS and harvested. Their DNA was isolated (NucleoSpin Blood XL, Macherey-Nagel) and prepared for PCR (Onestep PCR Inhibitor, Zymo Research) prior to processing for guide PCR amplification and sequencing by the Genetic Perturbation Platform (GPP) facility at the Broad Institute based on Broad recommended preparation protocols.

ORGANISM(S): Homo sapiens

PROVIDER: GSE179147 | GEO | 2022/04/14

REPOSITORIES: GEO