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RNA-guided systems, which employ complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, play a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors, like Cas9 and Cas12, perform guide RNA-dependent DNA cleavage. Although a few eukaryotic RNA-guided systems have been studied so far, such as RNA interference and ribosomal RNA modification, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided system (termed OMEGA) was reported. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins, raising the possibility that eukaryotes are also equipped with CRISPR-Cas/OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolved the structure of Spizellomyces punctatus Fz (SpuFz) at 2.7Ã… using cryogenic-electron microscopy, revealing the conservation of the core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life.