Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans

Abstract

Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.

Extended Data Fig. 1 |. piRNAi can silence a variety of germline-expressed genes.

a. Quantification of the number of self-progeny in C. elegans strains with sg-piRNAs targeting the sperm specific genes spe-8 and spe-12. Fertility was assayed from unmated L4 hermaphrodites in a him-5(e1490) mutant background. Control (‘−’) = randomized sg-piRNAs in cluster_A. Kruskal–Wallis ANOVA P = 0.0376, Dunn’s multiple comparison, * P = 0.0458, ns = P > 0.99. b. piRNAi against mes-4 and him-5 in wildtype (N2) animals. Transgenic animals were scored for sterile animals and for males (to identify ‘active arrays’). Two-tailed Mann-Whitney, * P = 0.0281 (sterility) and * P = 0.0455 (male frequency). c. piRNAi against dcr-1 and him-5 in wildtype animals (N2). Transgenic L4 stage animals were incubated at 20 °C and 25 °C and their progeny scored for lethality and males. dcr-1 genetic mutants are temperature-sensitive sterile at high temperatures. Two-tailed Mann-Whitney, ns P = 0.1320 and ** P = 0.0065. d. piRNAi against pie-1 and him-5 in the AID::PRG-1 strain maintained on 1 mM auxin (PRG-1 depleted). Larval stage animals from stable, independent transgenic lines were transferred to plates with or without auxin and scored for the number of progeny at 25 °C. Two-tailed Mann-Whitney, ** P = 0.0079. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) n = 4 (‘−’), n = 4 (spe-8), n = 3 (spe-12), (b) n = 6 (him-5), n = 5 (him-5 + mes-4), (c) n = 6 (all conditions), (d) n = 5 biologically independent transgenic strains.

Extended Data Fig. 2 |. Rules for efficient piRNA silencing.

a. Schematic of sg-piRNA cluster_E. b. Graph showing the effect of silencing him-5 with one, two, or three sg-piRNAs recoded in cluster_E. c. Bar graph showing the effect of silencing a codon-optimized gfp expressed in the germline (Pmex-5::gfp) with zero, one, two, three, or six sg-piRNAs from cluster_E. Independent biological strains (at least 11 animals per strain) were scored qualitatively on a dissection microscope blinded to genotype. d. Top. Schematic of the him-5 (D1086.4a.1) gene structure and the location of sg-piRNAs. Bottom. piRNAi using cluster_E targeting him-5 exons or 5’ and 3’ untranslated regions (UTRs) or introns (‘non-coding’). Control = randomized sg-piRNAs. e. Three versions of piRNA cluster_E using different sg-piRNAs were tested for him-5 silencing. Set 1 corresponds to Fig. 1b and the piRNAi transgenes in set 2 and set 3 target the same exons as set 1 but use different sg-piRNAs. f. The six sg-piRNAs in ‘set 1’ were shuffled (‘shuffle 1’ and ‘shuffle 2’), so each sg-piRNA was expressed by a different promoter in the piRNA cluster. The guide piRNA target locations in the him-5 transcript are shown as colored ovals. The strains were cultured at 25 °C. g. Transgenic animals were tested for him-5 silencing by piRNAi (piRNA cluster_E) over 12 generations at various temperatures (15 °C, 20 °C, 25 °C). h. Propagation of two transgenic animals with sg-piRNAs targeting him-5 (left) and him-8 (right) that initially had a low frequency of males in the population. The two strains were propagated for 12 generations, and the male frequency was quantified every six generations. i. C. elegans piRNA cluster_E targeting the gene cbr-him-8 in C. briggsae (AF16). Control (‘−’) = un-injected C. briggsae. Two-tailed t-test with Welch’s correction. ** P = 0.0236. Data are presented as mean values+/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (b) n = 5 (‘1’), n = 2 (‘2’), n = 4 (‘3’), n = 6 (‘4’), n = 4 (‘5’), n = 6 (‘6’), n = 4 (‘1 + 5’), n = 5 (‘2 + 4’), n = 4 (‘3 + 6’), n = 6 (‘1 + 4 + 5’), n = 4 (‘2 + 3 + 6’), (c) n = 4 (Neg. control), n = 6 (‘1’), n = 8 (‘2’), n = 10 (‘3’), n = 9 (‘4’), n = 7 (‘5’), n = 10 (‘6’), n = 8 (‘1 + 5’), n = 7 (‘3 + 4’), n = 5 (‘5 + 6’), n = 2 (‘2 + 3 + 6’), (d) n = 3 (control), n = 6 (Exons 2–4), n = 6 (exon 4), n = 5 (exons 5–6), n = 3 (spanning exons), n = 6 (5’ UTR), n = 6 (introns), n = 4 (3’ UTR), (e) n = 3 (set 1), n = 3 (set 2), n = 2 (set 3), (f) n = 3 (shuffle 1), n = 1 (shuffle 2), (g) n = 3 (all conditions), (h) n = 1 (all conditions), n = 3 (control), n = 3 (cbr-him-8) biologically independent transgenic strains.

Extended Data Fig. 3 |. piRNAi depends on plasmid structure and copy number.

a. Transgenic lines generated by injecting a piRNA cluster either as linear dsDNA (1.5 kb) or the same DNA cloned into a plasmid backbone (high copy ampicillin pTwist vector). The DNA transgenes were generated from cluster_E targeting him-5 and him-8 with six sg-piRNAs. Two-tailed Mann-Whitney tests. *** P = 0.0006, ns = not significant (0.0659). b. him-5 piRNAi plasmid (from panel a) digested with restriction enzymes that cuts in the bacterial vector backbone (left) or undigested (right) was used to induce silencing. The plasmid samples were treated identically (incubated in restriction enzyme buffer with or without restriction enzymes and purified over spin column) and injected at the same concentration. Two-tailed Mann-Whitney test. *** P = < 0.0001. c. Comparison of transgene copy number by whole genome sequencing of transgenic lines with extrachromosomal arrays formed from linear (green) or circular (purple) plasmids. d. Comparison of sg-piRNA expression targeting him-5 from transgenic strains carrying linearized and circular piRNAi transgenes. e. One ‘inactive’ multiplexed piRNAi strain (blue circle in Fig. 3h) was propagated for six generations and scored for males in the population and silencing of germline GFP fluorescence. ‘Generation 0’ corresponds to 2–3 generations after the initial injection. f. Whole-genome sequencing on the strain shown in panel e to determine the copy number of all plasmids in the extra-chromosomal array at the early (generation 1) and late (generation 6) timepoints. Mitochondrial DNA (mtDNA) copy number was used as a control. The copy number was calculated relative to the average sequencing coverage across the entire C. elegans genome. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) him-5: n = 8 (linear), n = 8 (circular), him-8: n = 7 (linear), n = 7 (circular), (b) n = 6 (digested), n = 6 (undigested), (c) n = 3 (all conditions), (d) n = 2 (linear), n = 2 (circular), (e) n = 1, (f) n = 1 biologically independent transgenic strains.

Extended Data Fig. 4 |. time-course of conditional cdk-1 silencing using auxin.

a. Brightfield image of animals with piRNAi targeting cdk-1 in the AID::PRG-1 strain on auxin (left panel) or off auxin (right panel). The dotted yellow line outlines fertilized embryos in the hermaphrodite uterus. Arrows indicate the vulva and embryos. cdk-1 encodes a cyclin-dependent kinase that is required for cell division and embryos arrest at the single-cell stage in cdk-1 mutants. Representative images from more than ten embryos imaged. Scale bar = 25 μm. b. piRNAi against cdk-1 in the AID:PRG-1 strain. Injected animals were maintained on 1 mM auxin plates (to deplete PRG-1). L4 stage animals were transferred to plates with or without auxin and surviving progeny was scored by first counting eggs and three days later counting the total number of adult progeny. Negative control = wildtype animals (N2). Kruskal-Wallis ANOVA P = 0.0036, Dunn’s multiple comparison * P = 0.0146, ns P = 0.3594. c. piRNAi activation after removal from auxin. To determine how long it takes to ‘turn on’ piRNA-mediated silencing after auxin removal, animals were transferred to non-auxin plates at each larval stage and at 3-hour intervals as young adults. The uteruses of adult animals (at least 11 animals) were scored for the presence of single-cell arrested embryos. Data are presented as mean values+/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (b) n = 3 (N2), n = 3 (auxin, both conditions), (c) n = 1 (all conditions) biologically independent transgenic strains.

Extended Data Fig. 5 |. piRNAi silencing tolerates up to three mismatches.

a. sg-piRNA mismatch tolerance using piRNA cluster_E. The six sg-piRNAs targeting him-5 each contained from zero to five mismatches. The schematics show the location of mismatches in the piRNA seed sequence (red boxes, nucleotides in positions 2 to 7) or in the remainder of the piRNA (white boxes, positions 8 to 21). For all sg-piRNAs (including controls), the leading ‘U’ was not modified as this base is required for piRNA transcription. In some transgenes the overall number of mismatches was constant but the location in each sg-piRNA was randomized (indicated by gray shading). The negative control contains inverted him-5 sg-piRNAs. b. Relationship between male frequency and the aggregate piRNA score calculated based on Wu et al. (2018), which takes the location of mismatch and wobble base pairing into account. Silencing data from panel a. Left: all piRNAs. Right: four highest expressed guide piRNA in cluster_E (the two remaining sg-piRNAs were rarely detectable by small RNA sequencing). Simple linear regression. R² = goodness of fit. c. Relationship between male frequency and the number of mismatches in each guide piRNA. Silencing data from panel a. Left: all piRNAs. Right: four highest expressed guide piRNA in cluster_E. Simple linear regression. R² = goodness of fit. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) Controls: n = 6, n = 6, n = 7, n = 8, n = 8, n = 5 (negative), n = 5, n = 9, n = 7, n = 7, n = 6, n = 6, n = 9 (positive), 1 mismatch: n = 9, n = 7, n = 8, n = 7, n = 6, n = 10 (technical and biological replicate), n = 7, n = 8, 2 mismatches: n = 5, n = 10, n = 8, n = 7, n = 6, n = 8, n = 6, n = 6, 3 mismatches: n = 7, n = 5, n = 8, n = 9, n = 5, 4 mismatches: n = 9, n = 6, n = 8, n = 8, n = 5, 5 mismatches: n = 7, n = 6, n = 7, n = 5, n = 7, n = 9, n = 8, n = 6, n = 9, n = 7, n = 5, n = 5, all values top to bottom, (b) n = 382, (c) n = 343 biologically independent transgenic strains.

Extended Data Fig. 6 |. Web application allows easy piRNAi transgene design.

a. Screenshot from www.wormbuilder.org/piRNAi. Simple mode to target a single gene with pre-defined criteria. b. Advanced mode. In the advanced mode, several different piRNA clusters can be re-coded with either custom 20-mers (for example, targeting gfp) or by selecting all 20-mers at a given edit distance mapping to a selected gene isoform. c. Output file with the piRNAi transgene annotated in GenBank format. The sequence is displayed in ‘A plasmid Editor’ (ApE).

Extended Data Fig. 7 |. Silencing endogenously gfp-tagged his-72 with piRNAi.

a. Brightfield and fluorescence images of a strain with an endogenously gfp-tagged his-72 locus. Images show a non-targeting control (mCherry), or sg-piRNAs targeting his-72, gfp, or his-72 + gfp. Images were acquired at 40x magnification using an oil immersion objective. Representative image from more than 50 adult animals imaged. White scale bar = 25 μm. b. We quantified GFP fluorescence in the nucleus of the ‘last’ three oocytes before the spermatheca using ImageJ (NIH). Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (b) n = 62 (N2), n = 59 (non-targeting), n = 64 (his-72), n = 62 (gfp), n = 62 (his-72 + gfp) fluorescent images from biologically identical transgenic strains across three technical replicates for each condition.

Extended Data Fig. 8 |. Amplification of short piRNAi transgenes from Matrix oligo Pools.

a. Schematic of two different 300 bp piRNAi transgenes, each encoding three sg-piRNAs. 300 bp oligos synthesized at large scale on oligo chips are delivered as arrayed sub-pools with 121 unique oligos in each of 384 wells. The piRNA transgenes are flanked by orthogonal forward and reverse primer sites that allow ‘dial-out’ PCR of transgenes from complex oligo pools (Kosuri et al., 2010). Each oligo pool contains 121 different oligos with a maximum length of 300 base pairs (Matrix Oligo Pools, Twist Bioscience). Two piRNAi transgenes (encoding six sg-piRNAs) are required for silencing. Each pool can, therefore, target 60 genes and individual piRNAi transgenes can be ‘dialed out’ using 16 orthogonal primers (8 forward * 8 reverse = 64 unique combinations). Restriction sites (DraI or EcoRV) allow Sanger sequencing of pair-wise amplified piRNA transgenes. b. 96-well amplification of 60 different piRNA transgene pairs targeting him-5 using two rounds of PCR. The first PCR was a bulk amplification of all oligos in the pool using all 16 amplification primers concurrently. 60 specific piRNA transgenes were amplified in a second round of PCR performed with pair-wise orthogonal primers listed above wells (expected size = 300 bp, indicated by arrow). Control reactions contained no template to assess background amplification of contaminants. With no optimization, we were able to amplify 51 of 60 piRNA transgenes (nine wells with weak or no band at 300 bp). Ladder = 100–10,000 bp VersaLadder (GoldBio). For a large-scale library (for example, a whole-genome library) a subcloning step after the first bulk amplification and transformation of the amplified oligo pool into bacteria would maintain long-term integrity and facilitate distribution of the oligo-pool as lyophilized plasmid pools. c. Representative example of Sanger sequencing of a PCR-amplified piRNA transgene from the oligo pool. 12 of 12 sequenced PCR products contained the expected three unique sg-piRNAs. From the sequencing trace, a low level of cross-talk is visible (minor peaks below the three sg-piRNAs), which can likely be minimized by reducing the number of PCR cycles and using a sub-cloned library as a PCR template.

Extended Data Fig. 9 |. Repressive chromatin modifications spreads in response to piRNAi.

a. ChIP-seq with antibodies against Pol II, H3K9me3, and H3K23me3 in two strains carrying piRNAi transgenes targeting him-5 (red trace) and him-8 (blue trace). The piRNAi target genes are indicated by dotted lines. zim-1, zim-2, zim-3, and him-8 are part of a single operon (CEOP4384, green bar). b. Control loci not targeted by piRNAi. c. mCherry and gfp were co-expressed in an operon under the mex-5 promoter (Pmex-5::mCherry::H2B - gpd-2 operon – gfp::h2b::cye-1 3’UTR) as a single copy insertion at ttTi5605 (Frøkjær-Jensen et al., 2008). The fluorophores were targeted individually or together (control) for silencing with piRNA transgenes using piRNA cluster_E. Data are presented as mean values+/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (c) mCherry: n = 3 (−/−), n = 5 (+/−), n = 7 (−/−), n = 5 (+/+), gfp: n = 3 (−/−), n = 5 (+/−), n = 7 (−/+), n = 5 (+/+) biologically independent transgenic strains.

Extended Data Fig. 10 |. piRNAi-induced transgenerational silencing.

a. Schematic showing assay to determine him-5 and him-8 inherited silencing. To quantify the proportion of males generated from self-fertilization (as opposed to male cross progeny generated by mating), we picked four to six unmated L4 animals in every generation to new plates and scored their progeny for males (out of 100 animals). The assay has a ‘transition generation’ (G₀) where a subset of animals are allowed to lose the extra-chromosomal piRNAi-array. Due to the shared germline cytoplasm, non-transgenic animals in the following G1 generation were exposed to sg-piRNAs in early embryonic development. The strains were not exposed to de novo sg-piRNAs generated during the L4 molt when sperm are made and female oocyte production is initiated. b. Duration of him-5 and him-8 transgenerational inheritance. We quantified transgenerational inheritance by counting the number of generations after losing the piRNA array before the fraction of males in the population was <= 1%. Data aggregated from panels Fig. 4bc and Supplementary Fig. 13. Kruskal-Wallis ANOVA, P=<0.0001, Dunn’s post hoc test *** P = 0.0007. c. Duration of transgenerational silencing in hrde-1 mutants. Two-tailed Mann-Whitney test, * P = 0.0286, ** P = 0.0079. d. Inherited silencing of piRNAi targeting spe-8 and spe-12 in a him-5(e1490) mutant background. Unmated L4 hermaphrodites were scored for viable progeny in the presence (solid black bar) or absence (solid white bar) of sg-piRNAs targeting spe-8 and spe-12, respectively. Control = him-5(e1490) animals with a randomized piRNAi cluster. Hygromycin was used to select transgenic animals, including controls, causing the increase in brood size of controls after the animals were grown on non-selective plates. Statistics: One-way ANOVA at each generation. * P = 0.0057 (generation −1), * P = 0.0127 (generation 0), ns P = 0.8312 (generation +1). Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (b) n = 6 (control), n = 14 (him-5), n = 8 (him-8), (c) him-5: n = 5 (N2), n = 5 (hrde-1), him-8: n = 4 (N2), n = 4 (hrde-1), (d) n = 2 (control), n = 2 (spe-8), n = 2 (spe-12) biologically independent transgenic strains.

Figure 1 –. Synthetic guide piRNAs silence endogenous genes.

a. Schematic overview of piRNA-mediated RNA interference (piRNAi). Synthetic guide piRNAs (sg-piRNAs, 21 nt) are expressed from semi-stable extra-chromosomal arrays and silence target mRNAs via transcriptional and post-transcriptional mechanisms. b. Silencing the endogenous him-5 gene with six sg-piRNAs expressed using 16 different recoded piRNA clusters (A to P) results in increased male frequency. The schematic above the him-5 locus indicates the location of antisense sg-piRNAs. Controls = him-5(e1490) mutant animals and negative controls: “1” = unmodified piRNA cluster_A, “2” = him-5 sense sg-piRNAs, “3” = six sg-piRNAs targeting gfp. Kruskal-Wallis ANOVA P < 0.0001, Dunn’s multiple comparison. *** P = <0.001 and n.s. P > 0.999. c. Gene silencing of him-5 or him-8 by RNAi by feeding (left), injection of in vitro transcribed dsRNA (middle), or piRNAi (right, cluster_E) in Pmex-5::gfp transgenic animals. Controls (“−”) = empty RNAi vector (pL4440), dsRNA targeting gfp, and piRNA cluster_E with randomized sg-piRNAs, respectively. Mann-Whitney test, comparison between RNAi and piRNAi condition. ** P = 0.0060 and * P = 0.0397. d. Male sperm expressing GFP from a Pmex-5::gfp transgene. The male germline is outlined with a white dotted line. 40x magnification. Scale bar = 25 um. e. Quantification of GFP expression in male sperm by visual inspection after silencing with RNAi (left) and piRNAi (right). Two-tailed t-test with Welch’s correction, ** P = 0.0049. f. sg-piRNAs targeting a ubiquitous gfp (Peft-3::gfp) silences fluorescence in the germline (not shown) and in embryos until approximately the 100-cell stage (visible GFP fluorescence indicated by white asterisks). Representative images from more than ten embryos imaged. Scale bar = 25 um. g. Conditional piRNAi using an auxin-inducible degron (AID) at the endogenous prg-1 locus. In the presence of auxin (“+”), PRG-1 is degraded. “−” = un-injected strain. Mann-Whitney test, * P = 0.0238, n.s. = not significant. h. Conditional piRNA-mediated silencing of cdk-1 in AID::PRG-1 animals result in single-cell embryonic arrest when transgenic animals are shifted to plates with no auxin. Arrowhead indicates the vulva. Representative images from more than ten adult animals. Scale bar = 25 um. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (b) n = 6 (him-5), n = 3 (“1”), n = 7 (“2”), n = 3 (“3”), n = 3 (A), n = 3 (B), n = 3 (C), n = 5 (D), n = 4 (E), n = 3 (F), n = 5 (G), n = 7 (H), n = 6 (I), n = 4 (J), n = 5 (K), n = 5 (L), n = 3 (M), n = 4 (N), n = 6 (O), n = 6 (P) biologically independent transgenic animals, (c) Feeding: n = 5 (all conditions) biologically independent animals; Injection: n = 10 (all conditions) biologically independent animals; piRNAi: n = 4 (“−”), n = 9 (him-5), n = 4 (him-8) biologically independent transgenic animals, (e) RNAi: n = 3 (both conditions) biologically independent animals, piRNAi: n = 3 (both conditions) biologically independent transgenic animals, (g) Wildtype: n = 3 (“−/−”), n = 5 (“−/+”), n = 3 (“+/+”), AID::PRG-1: n = 3 (“−/−”), n = 6 (“−/+”), n = 3 (“+/+”) biologically independent transgenic animals.

Figure 2 –. piRNAi tools for specific and scalable gene silencing.

a. Mismatch tolerance of piRNAi transgenes targeting him-5 containing zero to five mismatches in each of the six sg-piRNAs (see Extended Data Figure 5). Negative control = inverted sg-piRNAs. Kruskal-Wallis ANOVA P < 0.0001, Dunn’s multiple comparisons (control columnn “0 mismatch”), *** P = <0.001, * P = <0.032, and n.s. P >0.999 b. Screenshots from interactive online application (wormbuilder.org/piRNAi) to generate synthetic piRNAi transgenes. The app generates sequences that target endogenous genes in C. elegans or C. briggsae with pre-computed 20-mers containing at least four mismatches to any off-target genomic sequence. The app also allows the generation of custom piRNAi transgenes targeting, for example, a transgene or specific gene isoforms (Extended Data Figure 6). c. Two different short pairs of piRNAi transgenes (2× 300 bps) injected individually or together targeting him-5 or him-8. Kruskal-Wallis ANOVA, P < 0.0001, Dunn’s multiple comparison, *** P < 0.001, * P < 0.05, ns = not significant. d. Two pairs of short (300 bp) piRNA transgenes targeting gfp for silencing in a strain with GFP expression in germline (Pmex-5::gfp). Data are normalized to the total number of independent biological experiments (indicated above bars). Chi-square test on “No silencing” vs some effect (“Dim”, “Some silencing”, or “Silenced”) and corrected for multiple comparisons. *** P < 0.001, ** P = 0.006. e. Schematic of one piRNA transgene from a Matrix Oligo Pool (Twist Bioscience). 300 bp oligos synthesized at large scale on oligo chips are delivered as arrayed sub-pools with 121 unique oligos in each of 384 wells. The piRNAi transgenes include orthogonal primer sequences that allow selective amplification of any piRNAi transgene from the pool by “dial-out PCR”. f. PCR amplification of 2 × 60 piRNAi transgenes present in a single well (each 300 bp oligo encodes three sg-piRNAs). DNA ladder: 100–10,000 bp VersaLadder (GoldBio). All 12 individual piRNAi transgenes submitted for Sanger sequencing were correct (Extended Data Figure 8). Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) n = 358 biologically independent transgenic strains; individual data points show average male frequency for all strains injected with a given transgene containing mismatches: n = 6 (negative control), n = 7 (0 mismatches), n = 8 (1 mismatch), n = 8 (2 mismatches), n = 5 (3 mismatches), n = 5 (4 mismatches), n = 12 (5 mismatches) biologically independent experiments, (c) n = 14 (Neg. control), n = 12 (Pos. control), him-5: n = 6 (300 bp #1), n = 10 (300 bp #2), n = 4 (300 bp #1+2), him-8: n = 9 (300 bp #1), n = 12 (300 bp #2), n = 5 (300 bp #1+2) biologically independent transgenic animals, (d) n = 13 (Neg. control), n = 20 (Pos. control), n = 8 (300 bp #1), n = 7 (300 bp #2), n = 6 (300 bp #1+2) biologically independent transgenic animals.

Figure 3 –. piRNAi is specific and can be multiplexed.

a. Total number of piRNA reads mapping to endogenous or sg-piRNAs from cluster_A (centered on 21ur-199, purple) and the standard piRNA cluster (cluster_E, centered on 21ur-1224, green). See also Supplementary Figures 4–5. b. RNA-sequencing of secondary small RNAs (sRNA-seq) at the oma-1 locus in transgenic animals with piRNAi arrays targeting oma-1 or a control (unc-119). c. Pol II (top) and H3K9me3 ChIP-seq (bottom) in strains with sg-piRNAs targeting oma-1 (blue) or unc-119 (red). d. RNA sequencing (RNA-seq) of total RNA from young adult animals with piRNAi arrays targeting oma-1 (blue circle) or unc-119 (control, red circle). e. Comparison of mRNA expression of a mixed population of young adult animals (hermaphrodites and males) with piRNAi against him-5 and him-8 (black circles). f. Genetic requirements for silencing him-5 by piRNAi in N2 wildtype animals (green dots) or in mutant animals with primary effects on piRNAs (gray), P-granules (yellow), H3K9 methylation (red), nuclear RNAi (blue), and poly-UG tails (purple). Controls (black circles) are un-injected non-transgenic mutant strains. Mann-Whitney pairwise tests (injected vs un-injected). *** P < 0.0001, *** P < 0.001, ** P < 0.01, NS P > 0.05. g. Multiplexed piRNAi-mediated silencing of a temperature-sensitive oma-1 allele using arrays with one (oma-1) or four (him-5, gfp, oma-1, and mCherry) piRNAi transgenes. Positive control = N2 injected with oma-1 piRNAi, negative control = oma-1(zu405ts). Mann-Whitney tests, P *** = 0.0006, ns = not significant (P = 0.853). h. Multiplexed piRNAi against gfp and him-5 (in addition to oma-1 and mCherry) in transgenic Pmex-5::gfp animals. Neg. control = randomized sg-piRNAs. Purple and blue circles indicate matched data scored for males and GFP silencing in the same transgenic strain. Mann-Whitney test, P *** = 0.0001 (males) and *** = 0.0004 (GFP). i. sRNA-seq from active (GFP negative and high male frequency) and inactive (GFP expression and no males) piRNAi arrays in Pmex-5::gfp transgenic animals (no mCherry transgene present in strain). Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Negative controls = transgenic animals with randomized sg-piRNAs. Sense reads positive, antisense reads negative. Sample sizes (a) Cluster_A: n = 11 (endogenous), n = 2 (unc-119), n = 3 (oma-1), n = 1 (random), Cluster_A: n = 11 (endogenous), n = 3 (him-5), n = 2 (him-8), n = 1 (gfp), n = 1 (mCherry) small RNA libraries from biologically independent transgenic strains, (f) N2: n = 12 and n = 7, prg-1: n = 7 and n = 5, prde-1: n = 7 and n = 5, pgl-1: n = 6 and n = 7, mut-7: n = 5 and n = 7, znfx-1: n = 7 and n = 7, “met-2; set-25; set-32”: n = 5 and n = 7, set-25: n = 3 and n = 7, set-32: n = 3 and n = 7, met-2: n = 6 and n = 7, nrde-1: n = 3 and n = 7, nrde-2: n = 7 and n = 7, nrde-3: n = 3 and n = 7, hrde-1: n = 5 and n = 7, rde-3: n = 4 and n = 4 biologically independent transgenic strains (piRNAi and controls, respectively), (g) n = 7 (positive control), n = 6 (negative control), n = 6 (oma-1), n = 7 (multiplex) biologically independent transgenic strains, (h) n = 8 (negative control), n = 9 (multiplex) biologically independent transgenic strains.

Figure 4 –. piRNAi induces transgenerational silencing of endogenous genes.

a. Transgenerational inheritance of oma-1 silencing. oma-1(zu405ts) animals have a gain-of-function temperature-sensitive mutation inducing embryonic lethality at temperatures above 15°C (here, 20°C). Animals with oma-1 loss-of-function alleles are overtly normal, and oma-1 silencing results in viable animals at the non-permissive temperature. Transgenic strains with sg-piRNAs targeting oma-1 were established (G₋₂), allowed to lose the array (G₋₁ to G₀), and monitored for viable progeny in the absence of sg-piRNAs targeting oma-1 using an assay modified from Alcazar et al. (2008). Control = N2 with oma-1 sg-piRNAs. b. Inheritance of him-5 and him-8 silencing. Frequency of males in strains exposed to sg-piRNAs targeting him-5 or him-8 before and after the loss of the extra-chromosomal piRNAi-array (see also Extended Data Figure 10). Control = N2 animals with a piRNA cluster_A encoding randomized sg-piRNAs. c. Assays for inherited him-5 and him-8 silencing in a _nuclear heritable RNAi mutant (hrde-1). Control = N2 animals with piRNAi-arrays initially targeting him-5 or him-8. d. Inherited silencing of him-5 piRNAi is unaffected by auxin in wildtype (N2) animals. e. Inherited silencing of him-5 piRNAi becomes essentially permanent (RNA epigenetic) when PRG-1 is depleted by auxin. Arrows indicate transitions where animals are transferred between auxin and non-auxin plates leading to depletion and restoration of PRG-1, respectively. Dagger symbol indicates when AID::PRG-1 strains became sterile from the progressive mortal germline phenotype that is characteristic of prg-1 mutants. AID::PRG-1 animals have reduced levels of PRG-1 (approx. 20% of normal) in the absence of auxin. Data are presented as mean values +/− SEM with each data point corresponding to an independently derived transgenic strain. Sample sizes (a) n = 5 (control), n = 5 (oma-1), n = 4 (oma-1 multiplex), (b) n = 3 (control), n = 4 (him-5), n = 4 (him-8), (c) him-5: n = 3 (N2), n = 3 (hrde-1), him-8: n = 4 (N2), n = 4 (hrde-1), (d) n = 3 (no drug), n = 3 (on auxin), (e) n = 3 (all conditions) biologically independent transgenic strains. See Supplementary Figure 13 for biological replicates.