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.