GUIDEseq

GUIDE-seq, short for Genome-wide Unbiased Identification of DSBs Enabled by sequencing, is a method used to detect genome-wide double-strand breaks (DSBs) caused by programmable nucleases such as CRISPR-Cas9 in living cells. It provides an unbiased profile of on-target and off-target cleavage across the genome under specific experimental conditions.

The procedure involves delivering a nuclease complex (for example, Cas9 and a guide RNA) together with a short double-stranded oligodeoxynucleotide (dsODN) into cells. DSBs created by the nuclease are repaired by non-homologous end joining, during which the dsODN can become integrated at the break sites. Genomic DNA is then isolated, and sequences containing the dsODN junctions are enriched and sequenced. The resulting reads are mapped to the reference genome to identify the precise locations of DSBs, yielding a genome-wide map of cleavage sites and their relative frequencies.

GUIDE-seq is valued for its ability to reveal off-target activity without prior knowledge of potential off-target sites, helping researchers evaluate nuclease specificity and compare different nucleases or guide designs. It is particularly informative for assessing CRISPR-Cas9 variants and editing conditions in cell lines and, with limitations, in primary cells.

Limitations include dependence on efficient dsODN incorporation, possible bias toward readily accessible chromatin, and reduced sensitivity for very low-frequency off-target events. It also requires significant sequencing and computational analysis, and may not capture off-targets that do not efficiently integrate the dsODN. Despite these caveats, GUIDE-seq remains a foundational tool for genome-wide off-target profiling in gene-editing research.