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Cell-free DNA (cf.DNA) is a powerful noninvasive biomarker for cancer and prenatal testing, and it circulates in plasma as short fragments. To elucidate the biology of cf.DNA fragmentation, we explored the roles of deoxyribonuclease 1 (DNASE1), deoxyribonuclease 1 like 3 (DNASE1L3), and DNA fragmentation factor subunit beta (DFF with mice deficient in each of these nucleases. By analyzing the ends of cf.DNA fragments in each type of nuclease-deficient mice with those in wild-type mice, we show that each nuclease has a specific cutting p