APOBEC3A high-precision CRISPR-Cas9 base editors with minimized bystander and off-target mutations activity

Here, we describe an alternative a strategy for reducing base editor bystander mutations using a novel BE architecture that harbors an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN (R = A, G; V = G, A, C; Y = C, T) hierarchy. In direct comparisons with the widely used BE3 fusion in human cells, our eA3A-BE3 fusion exhibits comparable similar activities on cytidines in TC motifs but greatly reduced or no significant editing on cytidines in other sequence contexts. Importantly, Wwe show that eA3A-BE3 can corrects a human beta-thalassemia promoter mutation with much higher (>40-fold) precision than BE3, substantially minimizing the creation of an undesirable bystander mutation.. Surprisingly, Wwe also found demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.