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

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6 Scopus citations


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.
Original languageEnglish (US)
JournalG3 Genes|Genomes|Genetics
StatePublished - Jul 28 2022

Bibliographical note

KAUST Repository Item: Exported on 2022-09-14
Acknowledged KAUST grant number(s): OSR-CRG2020-4388
Acknowledgements: We thank Mohammed Aljohani for sharing unpublished reagents and Ramatoulaye Balde for excellent lab support. Research in C.F.-J.'s laboratory is supported by KAUST core funding and KAUST's Office of Sponsored Research (OSR-CRG2020-4388). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

ASJC Scopus subject areas

  • General Medicine


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