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Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac -targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology. [Display omitted] • A murine T cell-specific AAV variant, Ark313, is discovered through AAV6 evolution • A genome-wide CRISPR screen identifies QA2 an essential factor for Ark313 transduction • Ark313 enables efficient transgene delivery and targeting of large DNA payloads • Trac targeting of a CAR outperforms semi-random integration in a solid tumor model By combining structure-guided evolution and genome-wide screening, this work identifies an adeno-associated virus and co-factor that enable gene targeting at high efficiencies in mouse T lymphocytes, thus opening the path for T cell manipulations in murine models. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Pooled knockin of hundreds of TFs/surface receptors combined with different TCRs/CARs • Chronic stimulation screens discover programs to improve T cell persistence • Combinatorial knockin screens with ∼10,000 transcription factor combinations • BATF-TFAP4 dual knockin construct improves CAR-T cell fitness and function Modular pooled knockin screening (ModPoKI) is an adaptable platform that enables the evaluation of hundreds to thousands of different T cell constructs for engineered cellular immunotherapies. [ABSTRACT FROM AUTHOR]