An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities

Nat Biotechnol. 2018 Nov;36(10):977-982. doi: 10.1038/nbt.4199. Epub 2018 Jul 30.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems
  • Cytidine Deaminase / genetics*
  • DNA / genetics
  • Gene Editing / methods*
  • Genome
  • Humans
  • Mutation
  • Promoter Regions, Genetic / genetics
  • Proteins / genetics*
  • beta-Thalassemia / genetics


  • Proteins
  • DNA
  • CRISPR-Associated Protein 9
  • APOBEC3A protein, human
  • Cytidine Deaminase