Digenome-seq

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.

Advantages:

  • 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)

Disadvantages:

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


Reagents:

Illumina Library prep and Array Kit Selector



Reviews:

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



References:

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