Abstract 7234: The second-generation high-fidelity base editor AccuBaseTM (ceBE) can proficiently modify multiple genes within a human primary T cell while minimizing Off-target effects

Abstract Off target mutations, chromosomal rearrangements and Indels are the biggest limitation to the use of gene editing tools today. Base editing system provides an efficient method to modify the genome without double strand breaks (DSB). The biggest problem for base editing is the sgRNA-independent off-target. We utilize synthetic biology strategies to create a novel base editor AccuBaseTM (ceBE) that has the highest editing efficiency in the industry, with zero off target events. This was achieved by strategically inserting deaminases into Cas9 at specific, tolerance-verified sites, a process guided by an extensive transposon-based genetic screening, protein engineering, and direct evolution approach. AccuBaseTM protein forms a complex with the sgRNA, but it has not yet engaged with the target DNA and is not in contact with any non-targeting dsDNA. This mechanism avoids guide RNA independent indiscriminate off-target mutations that was a common problem of the first generation base editing tool. Upon guided by sgRNA, the AccuBaseTM protein associates with the target double-stranded DNA, triggering structural alterations within the AccuBaseTM protein. This results in the outward exposure of the deaminase domain, facilitating the editing of the target base. AccuBaseTM (ceBE) substantially reduces off-target effects while maintaining on-target editing when compared to BE4max. This improvement has been confirmed in multiple human cell lines, mouse cell lines, human primary T and NK cells, human embryonic stem cells, and in animal embryos. In this study, we apply AccuBaseTM to achieve multiplex gene modifications in primary human T cells, demonstrating its capacity for highly efficient multiplex gene disruption without inducing off-target effects. Importantly, the multiplex base-edited T cells exhibit enhanced ex-vivo cell expansion and do not exhibit the double-strand break-induced translocations observed in T cells edited with Cas9 nuclease. Citation Format: Han Ying, Guanglei Li, Liling Wang, Chunju Lu, Qi Lai, Chao Yao, Fenghua Lv, Shiguo Zhu, Tianhong Xu. The second-generation high-fidelity base editor AccuBaseTM (ceBE) can proficiently modify multiple genes within a human primary T cell while minimizing Off-target effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7234.