Reprogramming an endogenous small RNA pathway for multiplexed and transgenerational gene silencing in C. elegans

High-throughput sequencing analysis of C. elegans strains that were subject to piRNA-mediated RNA interference ("piRNAi"), a method developed in this study. The relative ease of targeting exogenous CRISPR/Cas proteins to specific DNA or RNA sequences using short guide RNAs has enabled the development of a wealth of tools in genetically tractable systems. Here, we demonstrate that an endogenous pathway can similarly be repurposed for gene silencing by expression of short guide piRNAs (21 nucleotides) in C. elegans. The cloning-free method, piRNA-mediated RNA interference ("piRNAi"), is specific, allows multiplexed gene silencing, and can silence genes in the female and male germline that are otherwise refractory to RNA interference (RNAi). Silencing requires several piRNAs expressed from extra-chromosomal DNA and tolerates up to four mismatches. Targeting decreases mRNA levels, increases secondary small interfering RNAs (22G), and results in repressive chromatin modifications at the target locus, indicating transcriptional and post-transcriptional silencing. We use piRNAi to induce transgenerational silencing of two canonical targets (gfp and oma-1) and identify two novel genes (him-5 and him-8) that inherit epigenetic silencing for four and six generations, respectively. small RNA-seq, mRNA-seq, and ChIP-seq of C. elegans that underwent piRNAi against different genes or control piRNAi