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While some eAgos have been demonstrated to use DNA guides for RNA cleavage, the ability of eAgos to cleave DNA targets remains unclear. In this study, we characterized CsAgo, an eAgo protein derived from thermophilic eukaryote Chaetomiumsp. MPI-CAGE-AT-0009, demonstrating a novel ability to cleave both DNA and RNA targets in vitro. Guided by short single-stranded DNA (ssDNA) or RNA, CsAgo exhibits robust RNA cleavage activity at 20\u201390\u00b0C in vitro.\u00a0CsAgo can effectively cleave ssDNA guided by RNA guides at 20\u201350\u00b0C in vitro. Notably, CsAgo can utilize DNA guides to effectively cleave ssDNA, plasmid double-stranded DNA (dsDNA), and linear dsDNA at\u00a0\u226580\u00b0C in vitro. Based on its ability to cleave dsDNA at high temperatures, CsAgo demonstrates versatility and efficacy in simplifying routine cloning workflows. Additionally, we have developed a CsAgo-based nucleic acid detection method based on a\u00a0Pyrococcus furiosus\u00a0Ago-mediated nucleic acid detection method, which exhibits a high sensitivity of six\u00a0copies\/reaction. These results suggest that eAgos include\u00a0that, in theory, can be utilized for potential DNA-targeting applications. This not only enhances our understanding of eAgos but also expands the toolkit for DNA manipulation.<\/jats:p>","DOI":"10.1093\/nar\/gkaf561","type":"journal-article","created":{"date-parts":[[2025,6,28]],"date-time":"2025-06-28T21:03:58Z","timestamp":1751144638000},"source":"Crossref","is-referenced-by-count":6,"title":["<i>In vitro<\/i> programmable DNA cleavage by a eukaryotic Argonaute"],"prefix":"10.1093","volume":"53","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-6490-4083","authenticated-orcid":false,"given":"Guangbo","family":"Yan","sequence":"first","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University , Wuhan , Hubei 430062 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xia","family":"Li","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key 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