Genome-wide profiling of adenine base editor specificity by EndoV-seq

Nat Commun. 2019 Jan 8;10(1):67. doi: 10.1038/s41467-018-07988-z.

Abstract

The adenine base editor (ABE), capable of catalyzing A•T to G•C conversions, is an important gene editing toolbox. Here, we systematically evaluate genome-wide off-target deamination by ABEs using the EndoV-seq platform we developed. EndoV-seq utilizes Endonuclease V to nick the inosine-containing DNA strand of genomic DNA deaminated by ABE in vitro. The treated DNA is then whole-genome sequenced to identify off-target sites. Of the eight gRNAs we tested with ABE, 2-19 (with an average of 8.0) off-target sites are found, significantly fewer than those found for canonical Cas9 nuclease (7-320, 160.7 on average). In vivo off-target deamination is further validated through target site deep sequencing. Moreover, we demonstrated that six different ABE-gRNA complexes could be examined in a single EndoV-seq assay. Our study presents the first detection method to evaluate genome-wide off-target effects of ABE, and reveals possible similarities and differences between ABE and canonical Cas9 nuclease.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Adenine / metabolism
  • Animals
  • Bacterial Proteins / metabolism*
  • Base Sequence / genetics
  • CRISPR-Associated Proteins / metabolism*
  • DNA / genetics
  • DNA / metabolism
  • Deamination
  • Deoxyribonuclease (Pyrimidine Dimer) / metabolism*
  • Fibroblasts
  • Gene Editing / methods
  • Genome, Human / genetics*
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Inosine / metabolism
  • Mice
  • Multiplex Polymerase Chain Reaction / methods
  • RNA, Guide, CRISPR-Cas Systems
  • Sensitivity and Specificity
  • Whole Genome Sequencing / methods*

Substances

  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • Inosine
  • DNA
  • Deoxyribonuclease (Pyrimidine Dimer)
  • Adenine