PIWI-interacting RNAs (piRNAs) play a crucial role in safeguarding genome integrity by silencing mobile genetic elements. From flies to humans, piRNAs originate from long single-stranded precursors encoded by genomic piRNA clusters. How piRNA clusters form to adapt to genomic invaders and evolve to maintain protection remain key outstanding questions. Here, we generate a roadmap of piRNA clusters across seven species that highlights both similarities and variations. In mammals, we identify transcriptional readthrough as a mechanism to generate piRNAs from transposon insertions (piCs) downstream of genes (DoG). Together with the well-known stress-dependent DoG transcripts, our findings suggest a molecular mechanism for the formation of piRNA clusters in response to retroviral invasion. Finally, we identify a class of dynamic piRNA clusters in humans, underscoring unique features of human germ cell biology. Our results advance the understanding of conserved principles and species-specific variations in piRNA biology and provide tools for future studies.

Department of Biochemistry University of Cambridge Tennis Court Road Cambridge CB2 1GA UK; Wellcome CRUK Gurdon Institute University of Cambridge Tennis Court Road Cambridge CB2 1QN UK

Department of Molecular Cellular and Developmental Biology Yale University New Haven CT 06511 USA; Yale Stem Cell Center Yale School of Medicine New Haven CT 06511 USA; Center for RNA Science and Medicine Yale School of Medicine New Haven CT 06511 USA

Institute for Physical Science and Technology University of Maryland College Park MD 20742 USA; Department of Physics University of Maryland College Park MD 20742 USA

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic; Bioinformatics Group Division of Molecular Biology Department of Biology Faculty of Science University of Zagreb 10000 Zagreb Croatia

National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD USA

National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD USA; Biophysics Graduate Program Institute for Physical Science and Technology University of Maryland College Park MD 20742 USA; Institute for Physical Science and Technology University of Maryland College Park MD 20742 USA; Department of Physics University of Maryland College Park MD 20742 USA

National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD USA; Oak Ridge Institute for Science and Education US Department of Energy Oak Ridge TN USA

National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD USA; Oak Ridge Institute for Science and Education US Department of Energy Oak Ridge TN USA; TriLab Bioinformatics Group National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD 20892 USA

National Institutes of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD USA; TriLab Bioinformatics Group National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda MD 20892 USA

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