An evolved AAV variant enables efficient genetic engineering of murine T cells

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Non-toxic delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knock-in efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knock-ins 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 pre-clinical 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 new avenues in experimental T cell immunology. Overall design: We performed a genome-wide CRISPR KO screen on pirmary murine T cells from C57BL6/J H11-Cas9 mice to identify essential host genes for newly discovered AAV serotype Ark313. Cells were transdcuced with the retroviral CRISPR library before being infected with Ark313 virus expressing GFP, cells were then sorted the cells based on GFP expression in to 4 bins (1-4, from low to high). Genomic DNA was exctracted and NGS analysis was performed on PCR amplicons from the genomic DNA.