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Updated: Jul 12, 2026
Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation
Published on: January 2, 2026
Single-cell genomics links targeted functional manipulations to efficacy-associated chromatin signatures in CAR T
James Scott-Browne1, Kole DeGolier1,2, Stephanie DeVoe1,2
1Department of Immunology and Genomic Medicine, National Jewish Health, Denver, CO, USA, 80206.
Abstract:
Chimeric antigen receptor (CAR) T cells produce extraordinary remission rates in some hematologic tumors, results inconsistently replicated across malignancies and/or target antigens. Anti-tumor efficacy can be enhanced by modifying CAR architecture or T cell differentiation state. Pooled screening methods to identify effects of modifications are often restricted to in vitro readouts of abundance or transcriptome, limiting ability to interrogate biology and project long-term T cell fates. We use genetically-encoded barcodes to track the effects of diverse functional manipulations on pooled murine and human CAR T cell chromatin states using scATAC-seq. We report stable and transient transcription factor activities programmed by cytokine concentration during in vitro expansion and altered in vivo effector differentiation programs driven by modifications to CAR antigen binding domain construction. These data establish genetic barcoding to tie targeted functional manipulations to single CAR T cell chromatin profiles in vitro and in vivo, providing insights toward augmented therapeutic efficacy.
Insights
Genetically-encoded barcodes reveal how modifying chimeric antigen receptor (CAR) T cell engineering impacts their chromatin state and long-term function. This method enhances understanding of CAR T cell biology for improved cancer immunotherapy.
Area of Science:
- Immunology
- Genetics
- Cancer Research
Background:
- Chimeric antigen receptor (CAR) T cells show promise in treating hematologic tumors but have inconsistent efficacy across different cancers.
- Enhancing anti-tumor activity requires optimizing CAR architecture and T cell differentiation, yet current screening methods are limited.
- Understanding long-term CAR T cell fate and biology is crucial for improving therapeutic outcomes.
Purpose of the Study:
- To develop and apply a genetic barcoding system for high-throughput analysis of CAR T cell modifications.
- To investigate the impact of functional manipulations on CAR T cell chromatin states using single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq).
- To link specific genetic modifications to CAR T cell biology and predict therapeutic potential.
Main Methods:
- Utilized genetically-encoded barcodes to track pooled murine and human CAR T cells undergoing diverse functional manipulations.
- Employed single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to analyze chromatin accessibility profiles.
- Correlated genetic modifications with CAR T cell chromatin states in vitro and in vivo.
Main Results:
- Identified stable and transient transcription factor activities influenced by cytokine concentration during in vitro expansion.
- Observed altered in vivo effector differentiation programs resulting from modifications in CAR antigen binding domain construction.
- Demonstrated the ability to link targeted functional manipulations to individual CAR T cell chromatin profiles.
Conclusions:
- Genetic barcoding coupled with scATAC-seq provides a powerful tool to dissect CAR T cell biology.
- Insights gained can inform strategies for engineering more effective CAR T cell therapies.
- This approach facilitates a deeper understanding of CAR T cell fate and function for augmented therapeutic efficacy.
