Answer to Question #178200 in Molecular Biology for Zohreh Khosravi

Question #178200

You performed a whole genome CRISPR screen in cancer cells to identify regulators of T cell killing. One of your hits leads to increased killing of your cancer cells in presence of T cells but it represents an uncharacterised protein. What is your hypothesis and which three experiments would you carry out to elucidate the function of your hit?


1
Expert's answer
2021-04-07T11:27:29-0400


Large-scale CRISPR screens have been challenging in primary human cells. We developed a new method, sgRNA lentiviral infection with Cas9 protein electroporation (SLICE), to identify regulators of stimulation responses in primary human T cells. Genome-wide loss-of-function screens identified essential T cell receptor signaling components and genes that negatively tune proliferation following stimulation. Targeted ablation of individual candidate genes validated hits and identified perturbations that enhanced cancer cell killing.

SLICE coupled with single-cell RNA-Seq revealed signature stimulation-response gene programs altered by key genetic perturbations. SLICE genome-wide screening was also adaptable to identify mediators of immunosuppression, revealing genes controlling response to adenosine signaling. The SLICE platform enables unbiased discovery and characterization of functional gene targets in primary cells.


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