Directed evolution of adenine base editors with increased activity and therapeutic application

The foundational adenine base editors (for example, ABE7.10) enable programmable A center dot T to G center dot C 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 protospacer adjacent motif (PAM) sites, ABE8s result in similar to 1.5x higher editing at protospacer positions A5-A7 and similar to 3.2x higher editing at positions A3-A4 and A8-A10 compared with ABE7.10. Non-NGG PAM variants have a similar to 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 gamma-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 messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.