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

Abstract

DNA single-strand breaks (SSBs), or 'nicks', are the most common form of DNA damage. Nicks occur at rates of tens of thousands per cell per day, and result from many sources including oxidative stress and endogenous enzyme activities. Accumulation of nicks, due to high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to learn how their locations and number 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 target sites. There is currently a pressing need for methods to study unintended nicking activity genome-wide to evaluate the impact of emerging genome editing tools on cells and organisms. Here we developed a new method, NickSeq, for efficient strand-specific profiling of nicks in complex DNA samples with single nucleotide resolution and low false-positive rates. NickSeq produces deep sequence datasets enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand. In this work, we apply NickSeq to profile off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. NickSeq will be useful in exploring the relevance of spontaneously occurring or repair-induced DNA breaks in human disease, DNA breaks caused by DNA damaging agents including therapeutics, and the activity of engineered nucleases in genome editing and other biotechnological applications.

CRISPR-Cas12a has widespread off-target and dsDNA-nicking effects

“…The unpredictable nature of the non-specific activated nicking makes it difficult to detect the outcomes of nicking. In addition, the commonly used methods to verify off-target editing do not detect nicks (31,32), meaning that detection of potential off-target effects due to non-specific nicking would require whole genome sequencing or specialized detection methods (58,59). Use of Cas12a orthologs, such as AsCas12a, that display reduced non-specific nickase activity may reduce these unpredictable effects during genome editing experiments.…”

High-throughputin vitrospecificity profiling of natural and high-fidelity CRISPR-Cas9 variants

“…It is also important to note that off-targets that can be nicked by Cas9 may also be sequestered by chromatin structure in vivo (Horlbeck et al, 2016;Yarrington et al, 2018). Nevertheless, new methods developed to detect nicks may be used to detect potential off-target nicking activity that are unreported in the previous studies (Cao et al, 2019;Elacqua et al, 2019).…”