Digenomeseq

Digenome-seq is a genome-wide, in vitro method for mapping nuclease-induced double-strand breaks, especially those created by CRISPR-Cas9, across the native genome. It is designed to identify off-target cleavage sites in a single experiment, providing a comprehensive view of nuclease specificity beyond targeted assays.

The method relies on purified genomic DNA that is incubated with a CRISPR-Cas9 complex containing a guide RNA. After the in vitro digestion, the DNA is prepared for whole-genome sequencing. Computational analysis looks for signatures of nuclease activity, such as characteristic ends or local changes in read distribution, to pinpoint both on-target and off-target cleavage sites across the genome.

Digenome-seq is used to assess the specificity of genome-editing nucleases, compare variants of Cas9 or other nucleases, and inform guide RNA design. Because it is performed in vitro, it does not require transfection into living cells and can reveal potential off-target sites that cellular assays might miss. It complements cell-based methods by offering a broader, genome-wide perspective on nuclease activity.

Advantages of the approach include its genome-wide scope and independence from cellular context, which can reveal rare or unexpected off-targets. Limitations involve reliance on high-quality reference genomes and deep sequencing, as well as the possibility that in vitro conditions do not fully replicate cellular environments. Consequently, results are often integrated with in vivo methods to provide a complete assessment of nuclease specificity.

Digenome-seq has been influential in the development of off-target detection workflows and is frequently cited alongside methods such as GUIDE-seq, SITE-seq, and CIRCLE-seq as part of CRISPR specificity studies.