A versatile one-shot high-multiplex base editing strategy enables the generation of 20-plex knockout CAR T cells with enhanced anti-tumor efficacy

The Mission Bio Tapestri platform allows for targeted single-cell DNA sequencing based on a microfluidic droplet technology. A custom Tapestri amplicon panel was designed to capture 56 potential ABE target regions across the human genome including the 20 distinct genes subject to viral vector-free, simultaneous high multiplex adenine base editing in primary human T cells in this study. The targeted genes are CD3E, B2M, CIITA, PVR, CD52, ICAM1, CD58, PDCD1, CTLA4, CD5, RASA2, MED12, ZC3H12A, RC3H1, TET2, ARID1A, SMARCC1, SMARCA4, SMYD3 and ADORA2A. This dataset reflects the single-cell level DNA editing efficiency of this 'single-shot' high efficiency editing approach in CAR transduced (anti-mesothelin CAR, M5) as well as untransduced primary human T cells at optimal dosing of the editing reagents. Furthermore, this dataset includes samples that underwent a dose titration of the 20-plex sgRNA mix.