Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID

Area of Science:

• Oncology
• Immunology
• Genetics

Background:

• Immunotherapies like PD-L1 blockade show limited efficacy in many patients.
• Pooled CRISPR-Cas9 knockout screens identified resistance genes in antigen presentation and cytokine signaling pathways.
• Arrayed CRISPRn screens offer potential for novel target identification and characterization in immuno-oncology.

Purpose of the Study:

• To develop and validate an arrayed CRISPRn screen in a tumor/immune co-culture system.
• To identify novel targets for immuno-oncology by investigating tumor resistance mechanisms.
• To characterize genes involved in immune synapse interactions and T cell killing.

Main Methods:

• Developed a small-scale arrayed CRISPRn screen using T cells and Cas9-expressing PC9 lung adenocarcinoma cells (PC9-OKT3 system).
• Designed a focused CRISPRn library targeting genes in antigen presentation, cytokine signaling, apoptosis, and immune synapse pathways.
• Assessed PC9 cell viability in 2D adherent co-cultures using longitudinal imaging analysis.

Main Results:

• Knockout of epidermal growth factor receptor (EGFR) and PLK1 increased tumor cell death in co-culture.
• Knockout of ICAM1 demonstrated subtle, donor-specific resistance to T cell killing.
• The study provides proof of concept for arrayed CRISPRn screens in tumor/immune co-culture models.

Conclusions:

• Arrayed CRISPRn screens are a viable tool for investigating tumor-immune interactions.
• This platform can identify novel targets for overcoming immunotherapy resistance.
• Further investigation of in vitro co-culture models is warranted for immuno-oncology advancements.