Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

Nat Biotechnol. 2017 Apr;35(4):371-376. doi: 10.1038/nbt.3803. Epub 2017 Feb 13.


Base editing induces single-nucleotide changes in the DNA of living cells using a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair. This genome editing approach has the advantage that it does not require formation of double-stranded DNA breaks or provision of a donor DNA template. Here we report the development of five C to T (or G to A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing 2.5-fold. Additionally, we engineered base editors containing mutated cytidine deaminase domains that narrow the width of the editing window from ∼5 nucleotides to as little as 1-2 nucleotides. We thereby enabled discrimination of neighboring C nucleotides, which would otherwise be edited with similar efficiency, and doubled the number of disease-associated target Cs able to be corrected preferentially over nearby non-target Cs.

MeSH terms

  • Bacterial Proteins / genetics*
  • Base Composition / genetics
  • CRISPR-Associated Protein 9
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Cytidine Deaminase / genetics*
  • Endonucleases / genetics*
  • Gene Editing / methods*
  • Genome / genetics*
  • Recombinant Fusion Proteins / genetics*


  • Bacterial Proteins
  • Recombinant Fusion Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • Cytidine Deaminase