Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity

Mol Cell. 2016 Aug 4;63(3):355-70. doi: 10.1016/j.molcel.2016.07.004.


Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance.

Publication types

  • Review
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Proteins / genetics
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems*
  • Computational Biology
  • DNA / genetics
  • DNA / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Gene Editing / methods*
  • Gene Targeting / methods*
  • Genomics / methods*
  • Humans
  • Kinetics
  • Mutation
  • Protein Engineering
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Substrate Specificity


  • Bacterial Proteins
  • CRISPR-Associated Proteins
  • RNA, Guide
  • DNA
  • Endonucleases