Quadruple adenine base edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease engineered T cells

Genome editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. For the SureSelect panel, baits were designed against all predicted on- and off-target regions in the human genome (hg38) for the gRNAs utilized in this study, based on sequence homology between the gRNA protospacer and the genome. Off-targets were defined as having up to five mismatches to any on-target or one indel and up to four mismatches to any on-target. This resulted in 15,920 captured regions for the Cas9 quadruple editing scenario and 25,988 captured regions for the ABE quadruple editing scenario. For targets with 2 mismatches, baits extended +/-100 bp around the cut site. Baits were tiled to predicted genomic targets to minimize binding within cut sites while maintaining optimal capture efficiency, irrespective of INDEL formation due to editing. For Cas9 and ABE, the cut site was assumed to be 3 bp upstream of the Protospacer Adjacent Motif (PAM). On-target sites and predicted off-targets with G edits within the ABE editing window (5-15 bp upstream of PAM). A site was considered edited if base edits exceeded 2% in ABE-edited samples while remaining <1% in controls. Translocation events were identified when paired reads mapped to different loci.