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Updated: Jul 16, 2025

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
Published on: November 13, 2021
Modular pooled discovery of synthetic knockin sequences to program durable cell therapies
Franziska Blaeschke1, Yan Yi Chen1, Ryan Apathy1
1Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
None:
Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions.

