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Updated: Apr 9, 2026
Dual CRISPR-Interference Strategy for Targeting Synthetic Lethal Interactions Between Non-Coding RNAs in Cancer Cells
Published on: May 30, 2025
High-content CRISPR activation screens identify synthetically lethal RNA-based mechanisms to sensitize cancer cells
Reece Villarin Akana1,2, Jeehyun Yoe1, Olivia Laveroni1
1Department of Genetics, Stanford University, Stanford, CA, USA.
Abstract:
T cells recognize their target cells through the T cell receptor (TCR). Combining gain-of-function, single-cell and optical high-content screens, we identified RNA-based mechanisms that selectively sensitize target cells to TCR-specific T cell cytotoxicity. First, CRISPR activation screens in melanoma cells identify functionally diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, WNT1 and others. Expressing sensitizing hits in cancer and virally infected cells restores TCR-specific cytotoxicity. Next, we developed in situ Perturb-seq for optical pooled genetic screens with in situ detection of perturbations and spatial transcriptomic readouts. Perturb-seq and in vivo-in situ Perturb-seq show that the hits converge on shared cell-autonomous and intercellular mechanisms, map gene-environment interactions and reveal that Wnt ligands activate T cells. Introducing a scalable approach to decode gene function at the cell and tissue level, the study uncovered context-specific gene functions to restore targeted T cell-based elimination of dysfunctional cells via synthetically lethal, RNA-based interventions.
Insights
This study identifies RNA-based strategies to enhance T cell-mediated killing of cancer cells. By uncovering new gene functions, researchers developed methods to restore targeted T cell therapies.
Area of Science:
- Immunology
- Genetics
- Cancer Biology
Background:
- T cells are crucial for adaptive immunity, recognizing targets via the T cell receptor (TCR).
- Enhancing T cell-mediated cytotoxicity is a key goal in cancer immunotherapy.
- Understanding genetic regulators of target cell sensitization is vital for improving T cell therapies.
Purpose of the Study:
- To identify RNA-based mechanisms that sensitize target cells to T cell-mediated killing.
- To uncover regulators of T cell receptor (TCR)-specific cytotoxicity.
- To develop scalable methods for decoding gene function in cellular and tissue contexts.
Main Methods:
- Utilized gain-of-function, single-cell, and optical high-content screening.
- Employed CRISPR activation screens in melanoma cells.
- Developed and applied in situ Perturb-seq for pooled genetic screens with spatial transcriptomics.
Main Results:
- Identified diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, and WNT1.
- Demonstrated that expressing sensitizing genes restores T cell-mediated killing in cancer and infected cells.
- Revealed convergence on cell-autonomous and intercellular mechanisms, including Wnt ligand activation of T cells.
Conclusions:
- Discovered context-specific gene functions to enhance T cell-mediated elimination of dysfunctional cells.
- Established RNA-based interventions for synthetically lethal approaches in T cell therapy.
- Provided a scalable platform for dissecting gene function at cellular and tissue levels for therapeutic applications.
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