Christian Frøkjær-Jensen

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 Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous 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 modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6–37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 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.

Despite the prominent role of endo-siRNAs in transposon silencing, their expression is not limited to these ‘nonself’ DNA elements. Transcripts of protein-coding genes (‘self’ DNA) in some cases also produce endo-siRNAs in yeast, plants and animals. How cells distinguish these two populations of siRNAs to prevent unwanted silencing of active genes in animals is not well understood. To address this question, we inserted various self-gene or gfp fragments into an LTR retrotransposon that produces abundant siRNAs and examined the propensity of these gene fragments to produce ectopic siRNAs in the Caenorhabditis elegans germline. We found that fragments of germline genes are generally protected from production of ectopic siRNAs. This phenomenon, which we termed ‘target-directed suppression of siRNA production’ (or siRNA suppression), is dependent on the germline expression of target mRNA and requires germline P-granule components. We found that siRNA suppression can also occur in naturally produced endo-siRNAs. We suggest that siRNA suppression plays an important role in regulating siRNA expression and preventing self-genes from aberrant epigenetic silencing. This article has an associated ‘The people behind the papers’ interview.