Froekjaer-Jensen, C. | bioRxiv Science

bioRxiv2022

rde-3 reduces piRNA-mediated silencing and abolishes inherited silencing in C. elegans.

Small RNA-mediated silencing of target genes can persist across generations and C. elegans is a well-established model for studying the molecular basis for epigenetic inheritance. We recently developed a piRNA-based inherited silencing assay that causes a high incidence of males by targeting him-5 and him-8. Acute gene silencing is determined in the presence of the piRNAi extra-chromosomal array and inherited silencing after loss of the piRNA trigger. This assay has the advantage of targeting endogenous genes that are easily scored in mutant backgrounds and obviates the need for mutant validation and genetic crosses, which can influence inherited silencing. Here we show an example of the assay by testing acute and inherited piRNA-mediated him-5 silencing in ribonucleotidyltransferase rde-3 (ne3370) mutant animals. In the absence of rde-3, acute silencing was reduced but still detectable, whereas inherited silencing was abolished.

genetics↗

bioRxiv2022

Modular safe-harbor Transgene Insertion (MosTI) for targeted single-copy and extrachromosomal array integration in C. elegans.

Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here we describe a modular safe harbor transgene insertion (MosTI) for use in C. elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by non-homologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25 - 75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7 - 44%) at MosTI landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for eight integrated arrays (6 to 37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of one array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

genetics↗