Related Experiment Video
Updated: Aug 14, 2026
In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Published on: November 2, 2020
In vivo genome-wide CRISPR screens of human T cells in solid tumours
Qi Liu1,2, Peixin Amy Chen3,4, Esha Urs3,4
1Department of Medicine, University of California San Francisco, San Francisco, CA, USA. qi.liu3@ucsf.edu.
Abstract:
Large-scale CRISPR screening in human T cells holds significant promise for identifying genetic modifications that enhance cellular immunotherapy. Yet, many regulators of T cell performance in solid tumours are not revealed in vitro1,2. In vivo screening in tumour-bearing mice is more physiological but has been limited by low intratumoural T cell recovery. Here we developed an in vivo model that efficiently recovers human T cells from solid tumours, permitting genome-wide CRISPR screens with few mice. Tumour-infiltrating T cells from this model exhibit hallmarks of dysfunction compared with splenic T cells, creating an ideal screening context. We performed two genome-wide CRISPR knockout screens to identify regulators of intratumoural T cell abundance and effector function. The abundance screen revealed the P2RY8-Gα13 GPCR signalling axis as a negative regulator of T cell tumour infiltration. The effector function screen identified GNAS as a key driver of T cell dysfunction in tumours, whose product, Gαs, acts as a convergent node downstream of multiple GPCRs sensing distinct suppressive ligands. Knockout of GNAS rendered T cells resistant to multiple suppressive cues and significantly improved efficacy across diverse solid tumour models in chimeric antigen receptor (CAR) and T cell receptor (TCR) systems. Combinatorial knockout of P2RY8-GNAS further enhanced tumour control, demonstrating that complementary in vivo screens can identify orthogonal targets whose combined editing improves therapeutic potency. This flexible, scalable platform can be adapted for systematic discovery of genetic strategies to improve solid tumour T cell therapies.
Insights
This study introduces a novel in vivo CRISPR screening model for human T cells in solid tumors. This model identifies genetic targets, like GNAS, to enhance T cell immunotherapy efficacy against cancer.
Area of Science:
- Immunology
- Genetics
- Cancer Biology
Background:
- CRISPR screening in human T cells can improve immunotherapy for solid tumors.
- Current in vitro models fail to identify key regulators of T cell function within the tumor microenvironment.
- Existing in vivo screening methods suffer from low T cell recovery from tumors.
Purpose of the Study:
- To develop a scalable in vivo model for genome-wide CRISPR screening of human T cells in solid tumors.
- To identify genetic regulators of T cell abundance and effector function within the tumor microenvironment.
- To discover novel targets for enhancing T cell-based cancer immunotherapies.
Main Methods:
- Developed a novel in vivo model for efficient recovery of human T cells from solid tumors in mice.
- Performed genome-wide CRISPR knockout screens to identify regulators of T cell infiltration and function.
- Utilized chimeric antigen receptor (CAR) and T cell receptor (TCR) systems to evaluate therapeutic efficacy.
Main Results:
- Identified the P2RY8-Gα13 GPCR signaling axis as a negative regulator of T cell tumor infiltration.
- Discovered GNAS as a key driver of T cell dysfunction, acting as a convergent node for suppressive GPCR signaling.
- Demonstrated that GNAS knockout enhances T cell resistance to suppressive cues and improves efficacy in solid tumor models.
- Showed that combined knockout of P2RY8 and GNAS further enhances anti-tumor activity.
Conclusions:
- The developed in vivo model enables efficient, large-scale CRISPR screening of T cells in solid tumors.
- Targeting GNAS and the P2RY8-Gα13 axis represents a promising strategy to enhance T cell immunotherapy for solid tumors.
- This platform facilitates the discovery of genetic modifications to improve T cell therapies for diverse solid cancers.
