Adenoviral vector DNA for accurate genome editing with engineered nucleases

Nat Methods. 2014 Oct;11(10):1051-7. doi: 10.1038/nmeth.3075. Epub 2014 Aug 24.


Engineered sequence-specific nucleases and donor DNA templates can be customized to edit mammalian genomes via the homologous recombination (HR) pathway. Here we report that the nature of the donor DNA greatly affects the specificity and accuracy of the editing process following site-specific genomic cleavage by transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nucleases. By applying these designer nucleases together with donor DNA delivered as protein-capped adenoviral vector (AdV), free-ended integrase-defective lentiviral vector or nonviral vector templates, we found that the vast majority of AdV-modified human cells underwent scarless homology-directed genome editing. In contrast, a significant proportion of cells exposed to free-ended or to covalently closed HR substrates were subjected to random and illegitimate recombination events. These findings are particularly relevant for genome engineering approaches aiming at high-fidelity genetic modification of human cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics*
  • Cell Line
  • Cell Separation
  • DNA / chemistry*
  • DNA, Viral / genetics*
  • Deoxyribonucleases / chemistry*
  • Gene Targeting / methods
  • Genetic Engineering / methods*
  • Genome
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Polymerase Chain Reaction
  • Recombination, Genetic
  • Repetitive Sequences, Nucleic Acid
  • Reproducibility of Results


  • DNA, Viral
  • DNA
  • Deoxyribonucleases