Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

Nat Biotechnol. 2023 May;41(5):673-685. doi: 10.1038/s41587-022-01533-6. Epub 2022 Nov 10.


Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Publication types

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

MeSH terms

  • Adenine
  • CRISPR-Cas Systems*
  • Cytidine / genetics
  • Cytosine*
  • DNA / genetics
  • Deoxyadenosines
  • Gene Editing
  • Humans


  • Cytosine
  • Adenine
  • DNA
  • Deoxyadenosines
  • Cytidine

Associated data

  • figshare/10.6084/m9.figshare.21210845