Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing

Mol Ther. 2016 Mar;24(3):475-87. doi: 10.1038/mt.2016.1. Epub 2016 Jan 11.


The rapid advancement in targeted genome editing using engineered nucleases such as ZFNs, TALENs, and CRISPR/Cas9 systems has resulted in a suite of powerful methods that allows researchers to target any genomic locus of interest. A complementary set of design tools has been developed to aid researchers with nuclease design, target site selection, and experimental validation. Here, we review the various tools available for target selection in designing engineered nucleases, and for quantifying nuclease activity and specificity, including web-based search tools and experimental methods. We also elucidate challenges in target selection, especially in predicting off-target effects, and discuss future directions in precision genome editing and its applications.

Publication types

  • Review

MeSH terms

  • Animals
  • Binding Sites*
  • CRISPR-Cas Systems
  • Computational Biology / methods*
  • Endonucleases / metabolism*
  • Gene Editing* / methods
  • Gene Targeting* / methods
  • Genome*
  • Genomics / methods*
  • Humans
  • Protein Binding
  • Web Browser


  • Endonucleases