Key Findings
The high-throughput in vivo CAR-T double-knockout (DKO) CRISPR screen identified multiple synergistic gene pairs that improve antitumour efficacy. The most effective combination identified is the NR4A1–SOCS3 DKO. This specific pair outperformed several benchmark single-gene and dual-gene modifications in both T cells and CAR-T cells.
Technical Details
Analysis via whole-transcriptome profiling and in vivo single-cell RNA sequencing indicates that NR4A1–SOCS3 DKO specifically upregulates metabolic pathways. This results in CAR-T cells with significantly higher mitochondrial activity and metabolic fitness. These modified cells exhibited potent antitumour efficacy and long-term tumour control across various tumour models without compromising safety features.
The study utilized several genomic resources for data analysis, including:
- Human reference genome GRCh38 Gencode v44 (bulk RNA-seq) and v37 (single-cell RNA-seq).
- Human reference genome GRCh38 Gencode v47 and ENCODE blacklist regions (WGS).
- Molecular Signatures Database (MSigDB v7.4) and the ProjecTILs single-cell T cell atlas dataset.
Data including CRISPR screen sequencing, bulk and single-cell RNA-seq, CUT&RUN, and WGS have been deposited in the Gene Expression Omnibus (GSE272887, GSE335215) and the Sequence Read Archive (PRJNA1477126).
Background & Context
While CAR-T cell therapy has transformed the treatment of haematological malignancies, its overall efficacy is limited by intrinsic negative regulatory mechanisms within T cells. This study addresses these limitations by employing a DKO screening approach to rapidly discover combination targets that can overcome these intracellular checkpoints.
Significance & Outlook
The identification of NR4A1–SOCS3 as a promising pair of intracellular checkpoints provides a pathway for engineering high-performance CAR-T therapies. By demonstrating the utility of high-throughput in vivo DKO screening, this research establishes a method for the rapid discovery of synergistic gene targets to enhance cancer immunotherapy.
Source: https://doi.org/10.1038/s41586-026-10956-z
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