High throughput sequencing of a single AAV mediated multiplexed activation of endogenous genes achieves aging rejuvenation and enhances antitumor immunity

Multiplexed genetic manipulations offer the potential to treat a variety of human diseases. Recent CRISPR-based transcriptional engineering systems present a promising therapeutic approach, but their use in vivo remains challenging. Here, we developed a compact dCasMINI based CRISPRa complex, miniCRISPRa, for efficient transcriptional activation at endogenous loci, allowing multiplexed gene activation via an all-in-one AAV vector. As proof of concept, we treated 22-month-old mice with a single dose of AAV-9 to activate Oct4, Sox2, and Klf4 for two months, achieving tissue rejuvenation and youthful transcriptomic states. Additionally, AAV-mediated miniCRISPRa enhanced the expression of endogenous interferons Ifna, Ifnb1, and Ifngamma in melanoma tumors, boosting antitumor immunity by increasing cytotoxic T and NK cell infiltration. Our study offers a powerful in vivo platform for gene modulation and therapies targeting a wide range of human diseases.