Cas9-Digested Whole-Genome Sequencing

Digenome-seq was designed to profile genome-wide Cas9 off-target effects (Kim et al., 2015). A multiplexed version has also been published (Kim et al., 2016). It belongs to a family of methods, including HTGTS (Chiarle et al., 2011), LAM-HTGTS (Hu et al., 2016), and Guide-seq (Kim et al., 2015), which are aimed at detecting off-target effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and other RNA-guided engineered nucleases (RGENs).

This method detects off-target mutations, induced by RGENs in a bulk population of cells, by sequencing in vitro nuclease-digested genomes (digenomes). These digests should produce many DNA fragments with identical 5_ ends, which are vertically aligned at cleavage sites.


  • Relies on DNA cleavage rather than binding
  • Performed in a genomic context and captures sites with a DNA/RNA bulge
  • Detects off-target effects with a frequency of 0.1% or lower (Mei et al., 2016)


  • Requires in vivo cleavage confirmation (Hu et al., 2016)
  • High skill requirement for bioinformatic analysis


Illumina Library prep and Array Kit Selector


Lee C. M., Cradick T. J., Fine E. J. and Bao G. Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Mol Ther. 2016;24:475-487

Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics. 2016;43:63-75


Kim D., Kim J., Hur J. K., Been K. W., Yoon S. H. and Kim J. S. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016;34:863-868