Applications of adenine base editors (ABEs) have been constrained by the limited compatibility of the deoxyadenosine deaminase component with Cas homologs other than SpCas9. We evolved the deaminase component of ABE7.10 using phage-assisted non-continuous and continuous evolution (PANCE and PACE), resulting in ABE8e. ABE8e contains eight additional mutations that increase activity (k app ) 590-fold compared with ABE7.10. ABE8e offers substantially improved editing efficiencies when paired with a variety of Cas9 or Cas12 homologs. ABE8e is more processive than ABE7.10, which could benefit screening, disrupting regulatory regions and multiplex base editing applications. A modest increase in Cas9-dependent and -independent DNA off-target editing, and in transcriptome-wide RNA off-target editing can be ameliorated by introducing additional mutations in the TadA-8e domain. Finally, we show that ABE8e can efficiently edit natural mutations in a GATA1 binding site in the BCL11A enhancer or the HBG promoter in human cells, targets which were poorly edited with ABE7.10. ABE8e broadens the effectiveness and applicability of adenine base editing.

We used BE-PACE to evolve new cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing GC, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width, and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.Genome editing has revolutionized the life sciences and entered clinical trials to treat genetic diseases. 1 The use of programmable nucleases to generate double-stranded DNA breaks (DSBs) followed by homology-directed repair can introduce a wide variety of modifications but is inefficient in non-dividing cells, and is typically accompanied by an excess of unwanted insertions and deletions (indels), translocations, or other chromosomal rearrangements. 2 Base editing directly modifies target DNA bases in living cells and has become widely used to correct or install point mutations in organisms ranging from bacteria to human embryos. 3 Base editors use a catalytically impaired Cas9 to open a single-stranded DNA loop at a specified genomic site (Fig. 1a). Bases within the editing window (typically ~5 nt wide) in this region are modified by a tethered base-modification enzyme that only accepts single-stranded DNA.