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DNA single-strand breaks (SSBs), or “nicks,” are the most common form of DNA damage. Oxidative stress, endogenous enzyme activities, and other processes cause tens of thousands of nicks per cell per day. Accumulation of nicks, caused by high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to characterize the mechanisms of these processes and learn how the location and number of nicks affect cells, disease progression, and health outcomes. In addition to natural processes, including DNA repair, leading genome editing technologies rely on nuclease activity, including nick generation, at specific target sites. There is currently a pressing need for methods to study off-target nicking activity genome-wide to evaluate the side effects of emerging genome editing tools on cells and organisms. Here, we developed a new method, DENT-seq, for efficient strand-specific profiling of nicks in complex DNA samples with single-nucleotide resolution and low false-positive rates. DENT-seq produces a single deep sequence data set enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand with single-base resolution at penetrance as low as one strand per thousand. We apply DENT-seq to profile the off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. DENT-seq will be useful in exploring the activity of engineered nucleases in genome editing and other biotechnological applications as well as spontaneous and therapeutic-associated strand breaks.

DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution