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

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…

• NS
  National Science Foundation
• HH
  Howard Hughes Medical Institute
• BA
  Bill and Melinda Gates Foundation
• HH
  Helen Hay Whitney Foundation
• HF
  Hertz Foundation
• DO
  Division of Intramural Research, National Institute of Allergy and Infectious Diseases

  Award: U01AI142756
• NI
  National Institutes of Health

  Awards: R01 AR063070, R35GM118062, NIH R01 AR063070, U01AI142756, RM1HG009490, R01AR063070, R01EB027793, NIH R01
• NH
  National Human Genome Research Institute

  Award: RM1HG009490
• NI
  National Institute of General Medical Sciences

  Award: R35GM118062
• NI
  National Institute of Diabetes and Digestive and Kidney Diseases

  Awards: NIH R01, U54DK106829
• NI
  National Institute of Biomedical Imaging and Bioengineering

  Awards: R01EB031172, R01EB027793