Circle-seq Method
Circle-seq Method - Related Videos
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FAQs
Circle-seq enriches cleavage-derived DNA by exploiting the different stability of circular and linear molecules. After genomic DNA is fragmented and circularized, exonuclease treatment removes remaining linear DNA, while circles persist. Rolling-circle amplification then increases the amount of surviving circular DNA for sequencing. This enrichment helps make nuclease-associated cut sites detectable across the genome.
Sequencing focuses on DNA fragments that survived selective processing and amplification as circles. When a nuclease cleavage event produces a fragment that can be retained and circularized, its sequence becomes enriched relative to uninformative linear DNA. Mapping these enriched sequences back to the genome identifies candidate cut locations, including unintended sites outside the intended target.
Because the workflow examines nuclease cleavage directly in fragmented genomic DNA, it does not require a cellular selection signal to reveal activity. This design can capture cleavage events that might not be represented through a cell-based selection process. Consequently, researchers can perform a broader genome-wide assessment of intended and unintended nuclease cutting.
An experiment begins by fragmenting genomic DNA and circularizing the resulting pieces. Exonuclease treatment removes residual linear DNA, leaving circles available for enrichment. Rolling-circle amplification generates amplified material from those survivors, and sequencing provides the readout used to locate nuclease-associated cleavage sites. The order is important because each stage prepares the DNA for the next enrichment step.
Genome-wide cleavage profiles can reveal whether a guide RNA directs cutting primarily to the intended site or also permits off-target cleavage. Those profiles help compare guide designs and characterize the targeting behavior of different CRISPR-Cas nucleases. The resulting information can support selection of editing reagents with a suitable specificity profile for a particular research application.
Circle-seq is particularly relevant when researchers need a broad specificity assessment for genome-editing applications with safety implications. By identifying unintended cleavage sites without requiring a cellular selection system, it provides evidence for evaluating nuclease behavior and guide choice. In therapeutic research, that information helps frame whether an editing strategy warrants further genome-wide safety assessment.