Transgenes can be permanently silenced in a single generation via a previously described small RNA-induced epigenetic silencing (RNAe) mechanism, which is promoted by the presence of a perfect Piwi-interacting RNA (piRNA) target site. In this study, we identify a previously unidentified mechanism capable of silencing single-copy transgenes that lack perfect piRNA target sites and that is triggered by a lack of chromosomal pairing during meiosis for multiple generations. Multigenerational RNAe can lead to reversible or permanent transgene silencing and may provide insight into variability in the expression of single-copy transgenes or single-copy genomic insertions, which are commonly used in experimental biology. Our analysis of “multigenerational RNAe” also offers new insights into potentially common epigenetic silencing events relevant to genome expression in the germline and embryo
