Genome-wide target specificities of CRISPR RNA-guided programmable deaminases

Nat Biotechnol. 2017 May;35(5):475-480. doi: 10.1038/nbt.3852. Epub 2017 Apr 10.


Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 ± 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.

MeSH terms

  • APOBEC-1 Deaminase / genetics
  • Bacterial Proteins / genetics*
  • Base Pairing / genetics
  • CRISPR-Associated Protein 9
  • CRISPR-Associated Proteins / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Cytidine Deaminase / genetics
  • Endonucleases / genetics*
  • Gene Editing / methods*
  • Genome, Human / genetics*
  • Humans
  • Mutagenesis, Site-Directed / methods*
  • Point Mutation / genetics
  • RNA / genetics*
  • Recombinant Fusion Proteins / genetics


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Recombinant Fusion Proteins
  • RNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases
  • APOBEC-1 Deaminase
  • APOBEC1 protein, human
  • Cytidine Deaminase