Digenome-Seq

Digenome-Seq

In vitro Cas9-digested whole-genome sequencing to profile genome-wide Cas9 off-target effects (Digenome-seq). A multiplexed version has also been published. It belongs to a family of methods such as HTGTS, LAM-HTGTS, and Guide-seq that are aimed at detecting off-target effects of 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 give rise to sequence reads that are vertically aligned at cleavage sites.

Pros:

• Relies on DNA cleavage rather than binding
• Performed in a genomic context and sites with a DNA/RNA bulge are captured
• Off-targets can be detected with a frequency of 0.1% or lower¹

Cons:

• Requires in vivo cleavage confirmation
• High skill requirement for bioinformatic analysis²

• Digenome-Seq: Kim D., Bae S., Park J., Kim E., Kim S., et al. (2015) Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods 12: 237-243, 231 p following 243
• 1. Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. (2016) Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics 43: 63-75
• 2. Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W., et al. (2016) Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11: 853-871

1. Kim D., Kim J., Hur J. K., Been K. W., Yoon S. H., et al. (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol
2. Lee C. M., Cradick T. J., Fine E. J. and Bao G. (2016) Nuclease Target Site Selection for Maximizing On-target Activity and Minimizing Off-target Effects in Genome Editing. Mol Ther 24: 475-487
3. Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. (2016) Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics 43: 63-75