Base editor technology, which uses CRISPR-Cas9 to direct cytidine deaminase enzymatic activity to specific genomic loci, enables the highly efficient introduction of precise cytidine-to-thymidine DNA alterations. However, existing base editors create unwanted C-to-T alterations when more than one C is present in the enzyme's five-base-pair editing window. Here we describe a strategy for reducing bystander mutations using an engineered human APOBEC3A (eA3A) domain, which preferentially deaminates cytidines in specific motifs according to a TCR>TCY>VCN hierarchy. In direct comparisons with the widely used base editor 3 (BE3) fusion in human cells, our eA3A-BE3 fusion exhibits similar activities on cytidines in TC motifs but greatly reduced editing on cytidines in other sequence contexts. eA3A-BE3 corrects a human β-thalassemia promoter mutation with much higher (>40-fold) precision than BE3. We also demonstrate that eA3A-BE3 shows reduced mutation frequencies on known off-target sites of BE3, even when targeting promiscuous homopolymeric sites.

The foundational adenine base editors (e.g. ABE7.10) enable programmable C•G to T•A point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG PAM sites, ABE8s result in ~1.5x higher editing at protospacer positions A5-A7 and ~3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2, with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98-99% target modification which is maintained when multiplexed across three loci. Delivered as mRNA, ABE8s induce no significant levels of sgRNA-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. Here we purified A3A (N57Q)-BE3 protein for ribonucleoprotein (RNP) electroporation of human peripheral blood (PB) mobilized CD34 + hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base