Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases

Biotechnol Adv. 2017 Jan-Feb;35(1):95-104. doi: 10.1016/j.biotechadv.2016.12.003. Epub 2016 Dec 21.

Abstract

The development of customizable sequence-specific nucleases such as TALENs, ZFNs and the powerful CRISPR/Cas9 system has revolutionized the field of genome editing. The CRISPR/Cas9 system is particularly versatile and has been applied in numerous species representing all branches of life. Regardless of the target organism, all researchers using sequence-specific nucleases face similar challenges: confirmation of the desired on-target mutation and the detection of off-target events. Here, we evaluate the most widely-used methods for the detection of on-target and off-target mutations in terms of workflow, sensitivity, strengths and weaknesses.

Keywords: CRISPR; Cas9; Engineered nucleases; Genome editing; Indel detection; Mismatch detection assays; Unbiased off-target analysis.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Deoxyribonucleases* / genetics
  • Deoxyribonucleases* / metabolism
  • Gene Editing / methods*
  • Humans
  • Mice
  • Mutation / genetics*

Substances

  • Deoxyribonucleases