C. elegans is a convenient model for studying small RNA-mediated inherited silencing due to the animal's short generation time (three days) and the ability to identify molecular pathways in genetic screens (Burton et al., 2011; Buckley et al., 2012; Spracklin et al., 2017). Epigenetic silencing of an endogenous gene is often done by targeting a temperature-sensitive gain-of-function allele of
oma-1(
zu405) with dsRNA, and silencing persists for up to three generations (Alcazar et al., 2008). For a visual read-out, single-copy transgenes with GFP expression in the germline (Zeiser et al., 2011; Frkjr-Jensen et al., 2012; Nance and Frkjr-Jensen, 2019) have been engineered to contain endogenous piRNA binding sites in the 3' UTR (Ashe et al., 2012; Lee et al., 2012; Shirayama et al., 2012; Bagijn et al., 2012). For transgenes, piRNA-induced silencing persists longer and sometimes indefinitely. Genetic factors required for small RNA-mediated inherited silencing have primarily been identified by crossing silenced piRNA gfp sensor strains into mutant genetic backgrounds (Ashe et al., 2012; Lee et al., 2012; Shirayama et al., 2012; Luteijn et al., 2012). However, introducing mutations by genetic crosses raises several concerns. First, there are several examples of mating causing changes in epigenetic inheritance. For example, the lack of transgene pairing during meiosis after a cross can lead to permanent transgene silencing via PRG-1-dependent mechanisms (Leopold et al., 2015), and mating can induce multigenerational silencing inherited for over 300 generations (Devanapally et al., 2021). Moreover, Dodson and Kennedy (2019) characterized a transgenerational disconnect between the genotype and phenotype (sensitivity to exogenous RNAi) of
meg-3/4 mutants for more than seven generations after a genetic cross. Second, crosses frequently require molecular genotyping, which makes it cumbersome to perform many biological replicates. Third, there are some concerns about using transgenes as a proxy for endogenous gene silencing. For example, most piRNA sensor strains include synthetic piRNA binding sites in the 3' UTR (Ashe et al., 2012; Lee et al., 2012; Shirayama et al., 2012; Bagijn et al., 2012), but endogenous genes are resistant to piRNA silencing when targeting their 3' UTRs (Priyadarshini et al., 2022; Wu et al., 2022). Moreover, transgene insertion site (Frkjr-Jensen et al., 2014), non-coding DNA structures (Frkjr-Jensen et al., 2016), coding sequence (Fielmich et al., 2018; Aljohani et al., 2020), and transgene structure (El Mouridi et al., 2022) can influence epigenetic silencing. These observations suggest that transgenes may not fully recapitulate the balance between silencing foreign DNA and protecting endogenous gene expression (Frkjr-Jensen, 2019). Finally, distinguishing between silencing initiation and maintenance phases is complicated using genetic crosses. Experiments require crossing mutant alleles to sensor strains, de-repress silencing, and outcrossing mutations to monitor de novo establishment of silencing (Shirayama et al., 2012).