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Updated: Aug 6, 2026
Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation
Published on: January 2, 2026
High-throughput single-cell screening of a cross-lineage CAR library for early-stage CAR design and candidate
Hairong Jing1, Dan Yuan1, Bangquan Ye1
1BOE Technology Group Co. Ltd., Beijing, China.
Abstract:
Chimeric antigen receptor T cell therapy (CAR-T) has demonstrated promising efficacy in hematological malignancies, but translating that success to solid tumors remains challenging. Here, we construct a CAR library comprising approximately 1000 variants targeting prostate-specific membrane antigen by recombining transmembrane (TM), co-stimulatory, and activation domains from Natural Killer (NK) and T cell receptors. Single-cell screening identifies ICOSTM-containing variants with improved T cell activation; NK-derived activation domains, such as DAP10ζ, DAP12ζ, and FcRγζ, further augment the effector capacity. Gene regulatory network analysis reveals that CAR variants with elevated expression of T cell-activation-related transcription factors correlates with enhanced cell function. Overall, our study advances early-stage CAR design by expanding the repertoire of structural components from diverse immune cells, providing a scalable platform for identifying candidates with functional profiles comparable to clinical benchmarks.
Insights
Researchers developed novel chimeric antigen receptor T cell (CAR-T) therapies by combining components from T and Natural Killer (NK) cells. This approach enhances CAR-T cell function for potential use against solid tumors like prostate cancer.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptor T cell (CAR-T) therapy shows success in blood cancers but faces challenges in solid tumors.
- Prostate-specific membrane antigen (PSMA) is a target for CAR-T therapy in prostate cancer.
Purpose of the Study:
- To engineer novel CAR-T variants with improved efficacy for solid tumors.
- To explore the recombination of transmembrane, co-stimulatory, and activation domains from T and Natural Killer (NK) cells.
Main Methods:
- Constructed a library of approximately 1000 CAR variants targeting PSMA.
- Utilized single-cell screening to identify promising CAR variants.
- Analyzed gene regulatory networks to understand CAR function.
Main Results:
- Identified CAR variants containing ICOS transmembrane domains that enhance T cell activation.
- NK-derived activation domains (DAP10ζ, DAP12ζ, FcRγζ) further improved effector function.
- Elevated expression of T cell-activation transcription factors correlated with enhanced CAR function.
Conclusions:
- Expanded the repertoire of CAR structural components by incorporating elements from diverse immune cells.
- Developed a scalable platform for identifying potent CAR candidates for solid tumors.
- Demonstrated the potential of novel CAR designs to achieve functional profiles comparable to clinical benchmarks.
