Genome-wide CRISPRa screens identify modulators of CAR T survival across diverse tumor cytokine milieus.
5 affiliations
- 1Seattle Children's Therapeutics, Seattle Children's Research Institute, 1920 Terry Ave, Seattle, WA 98101, USA.
- 2Department of Laboratory Medicine and Pathology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA.
- 3Ben Towne Center for Childhood Cancer and Blood Disorders Research, Seattle Children's Research Institute, 1920 Terry Ave, Seattle, WA 98101, USA.
- 4Department of Bioengineering, University of Washington, 3720 15th Ave NE, Seattle, WA 98195, USA.
- 5Division of Hematology, Oncology, and Bone Marrow Transplant, Department of Pediatrics, University of Washington Medical School, 4800 Sand Point Way NE, Seattle, WA 98105, USA.
Abstract
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies, and extending this success to other cancers remains an urgent clinical objective. CRISPR activation (CRISPRa) is a gene nomination platform with the potential to direct next-generation cell product development, but genome-wide surveys using this technology in primary T cells have been limited by prohibitive cell requirements and short-lived activity. Here, we describe a CAR-restricted T cell expansion platform generating yields of CRISPRa-CAR T cells far exceeding those required for genome-scale screening (∼10⁹). CRISPRa activity is sustained by incorporating divergent, duplicate elongation factor 1α core/human T cell leukemia virus hybrid promoters and by delivering guide RNAs late by lentiviral transduction to avoid losing perturbations that compromise early T cell activation or expansion. Manufactured CRISPRa-CAR T cells maintain potent on-target gene overexpression (>85% target⁺) across varied cell subsets, including those associated with T cell exhaustion, and over extended culture periods. When applied to survival-based genome-wide screens, CRISPRa-CAR T cell pools nominate endogenous genes that enhance CAR T cell persistence across varied cytokine levels in the presence of tumor (e.g., IRF4, GFI1, and GSE1). In a proof-of-concept study, we find that the survival effect of GSE1 can be preserved using a clinically relevant transgene overexpression strategy. More broadly, this screening framework establishes a foundation for systematic identification and downstream validation of genes that enhance cellular therapies.
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BibTeX
@article{curtis2026genomewide,
title = {{Genome-wide CRISPRa screens identify modulators of CAR T survival across diverse tumor cytokine milieus.}},
author = {Benjamin C Curtis and Cailyn H Spurrell and Lindsay Flint and Aalton M Lande and Marissa Leonardi and James M Rosser and Tavie Parker and Tiffanie Chai and Ardizon Valdez and Ryma Toumi and Nat Murren and Michael Fitzgerald and Jasmin Martinez-Reyes and Finn Watt and Christopher P Saxby and Corinne Summers and Kamila S Gwiazda and Michael C V Jensen and Shannon K Oda},
journal = {Molecular therapy. Nucleic acids},
year = {2026},
month = sep,
doi = {10.1016/j.omtn.2026.103117},
url = {https://europepmc.org/article/MED/42927201},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Genome-wide CRISPRa screens identify modulators of CAR T survival across diverse tumor cytokine milieus.
AU - Benjamin C Curtis
AU - Cailyn H Spurrell
AU - Lindsay Flint
AU - Aalton M Lande
AU - Marissa Leonardi
AU - James M Rosser
AU - Tavie Parker
AU - Tiffanie Chai
AU - Ardizon Valdez
AU - Ryma Toumi
AU - Nat Murren
AU - Michael Fitzgerald
AU - Jasmin Martinez-Reyes
AU - Finn Watt
AU - Christopher P Saxby
AU - Corinne Summers
AU - Kamila S Gwiazda
AU - Michael C V Jensen
AU - Shannon K Oda
PY - 2026
DA - 2026/09/28
JO - Molecular therapy. Nucleic acids
DO - 10.1016/j.omtn.2026.103117
UR - https://europepmc.org/article/MED/42927201
AB - Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies, and extending this success to other cancers remains an urgent clinical objective. CRISPR activation (CRISPRa) is a gene nomination platform with the potential to direct next-generation cell product development, but genome-wide surveys using this technology in primary T cells have been limited by prohibitive cell requirements and short-lived activity. Here, we describe a CAR-restricted T cell expansion platform generating yields of CRISPRa-CAR T cells far exceeding those required for genome-scale screening (∼10⁹). CRISPRa activity is sustained by incorporating divergent, duplicate elongation factor 1α core/human T cell leukemia virus hybrid promoters and by delivering guide RNAs late by lentiviral transduction to avoid losing perturbations that compromise early T cell activation or expansion. Manufactured CRISPRa-CAR T cells maintain potent on-target gene overexpression (>85% target⁺) across varied cell subsets, including those associated with T cell exhaustion, and over extended culture periods. When applied to survival-based genome-wide screens, CRISPRa-CAR T cell pools nominate endogenous genes that enhance CAR T cell persistence across varied cytokine levels in the presence of tumor (e.g., IRF4, GFI1, and GSE1). In a proof-of-concept study, we find that the survival effect of GSE1 can be preserved using a clinically relevant transgene overexpression strategy. More broadly, this screening framework establishes a foundation for systematic identification and downstream validation of genes that enhance cellular therapies.
SN - 2162-2531
ER -