Digital Counting of Breaks Labeling In Situ: A Fast and Absolute Quantification Method for Measurement of DNA Double-Strand Breaks Based on Digital Polymerase Chain Reaction

Abstract

DNA double-strand break (DSB) is the most dangerous type of DNA damage. In addition, DSBs are also common consequences of various therapeutic and genetic modifications. Therefore, quantification of DSB is of great importance in many fields including DNA damage repair, cancer therapy, gene editing, and radiation biology. Current methods are either low-throughput, laborious, or high cost. Here, we developed dc-BLIS (digital counting of breaks labeling in situ), a new method that can rapidly and precisely quantify the number of intracellular DSBs at a low cost by digital polymerase chain reaction. Using dc-BLIS, we quantified and compared the amount of DSBs induced by anti-cancer drugs, Cas9 variants, and different radiation doses, proving the capacity of dc-BLIS to quantify DSBs. We propose that dc-BLIS is suitable for various application scenes that require rapid and precise quantification of DSBs, including drug screening, gene-editing tool modification, and radiation effect assessment.

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“… 7 , 8 Moreover, the overload of DNA repair systems is unlikely, as the capacity of DNA repair machinery is very high; hundreds of DSBs per cell are repaired when induced by irradiation, laser beams, or chemical treatment. 38 For understanding of the phenomenon of DNA degradation even upon a single nucleolytic cleavage, it is important to note the kinetics and repair of Cas9-induced DSBs. In contrast to physiological DSBs, DNA damage response factors are not recruited to Cas9-induced DSBs immediately because Cas9 stays tightly bound to one or both DNA ends.…”