Tools for experimental and computational analyses of off-target editing by programmable nucleases

Nat Protoc. 2021 Jan;16(1):10-26. doi: 10.1038/s41596-020-00431-y. Epub 2020 Dec 7.


Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • Genomics / methods
  • High-Throughput Nucleotide Sequencing / methods
  • Humans


  • Endonucleases