Reprogramming deaminase substrate specificity for single nucleobase editing

In this study, we set out to reprogram deaminase context specificity to pinpoint editing. We identified multiple nucleic acid-recognition hotspots in the E. coli tRNA-specific adenosine deaminase (TadA). Strategically sampling these recognition hotspots, we first accessed multipotency for C in TadA and subsequently eliminate its A-deamination activity. We further reprogrammed TadAC context specificity through 16 evolution campaigns, each aimed at a defined NCN context, and isolated hundreds of thousands of context-specific cytosine deaminases. Our panel of 16 NCN-specific deaminases covers the full spectrum of all possible minus1 and plus 1 contexts for a target C, offering on demand deaminase choices for editor customization. Our context-specific CBEs corrected 5,866 of 7,196 disease-associated T:A-to-C:G transitions documented by ClinVar with higher accuracy than existing CBEs, often achieving selective editing of a single cytosine out of multiple cytosines in the protospacer without compromising editing potency. We also showcased the application of context-specific base editing for modeling disease-associated C:G-to-T:A transitions using two cancer driver mutations, KRASG12D and TP53R248Q, each demanding selective editing of one cytosine in two consecutive cytosines (ACC and CCG). These context-specific editors, as expected, showed tightly controlled off-target profiles by rejecting most cytosines at potential off-target sites. Bystander-free, single-nucleobase editing, as enabled by reprogramming deaminase context specificity, complements our current editor portfolio and unlocks new potential in base editing. Various CBE variants was transfected to cells which harbors 12,000 Disease-related-A paired sgRNA-target library. After 72h, genomic DNA was extracted and target was amplified using pcr. Amplicon library was submitted to Nextseq 2000 for sequencing.