CHANGEseq

CHANGE-seq is a high-throughput, in vitro method for genome-wide mapping of CRISPR-associated nuclease off-target cleavage sites. It is designed to evaluate potential off-target activity of guide RNAs and nuclease variants before applying editing in living cells. By using purified genomic DNA as a substrate and sequencing-based readouts, CHANGE-seq profiles where a CRISPR nuclease is likely to cut.

The workflow centers on capturing nuclease-induced cleavage events in a circularized DNA library. Genomic DNA is exposed to a CRISPR nuclease complex guided by a specified sgRNA, producing double-strand breaks at on-target and off-target loci. The resulting DNA ends are processed to generate circular DNA molecules that mark cleavage sites. After exonuclease treatment removes undigested fragments, the circular substrates are linearized and prepared for high-throughput sequencing. Sequencing reads are mapped to the reference genome to identify and quantify cleavage events, yielding a genome-wide map of potential off-target sites and relative cleavage frequencies.

CHANGE-seq is valued for its sensitivity and scalability, enabling comparison of different guide RNAs, nuclease variants, and experimental conditions in a single assay. It complements in vivo methods by providing a broad, sequence-level view of off-target potential without cellular context, which can inform guide design and safety assessments.

Limitations include its in vitro nature, which may not fully reflect chromatin structure and cellular repair processes that influence editing in living cells. Results should be interpreted alongside in vivo data and other off-target detection methods.