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

Abstract

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.

High-Throughput Library Transgenesis inCaenorhabditis elegansvia Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

“…However, the focus of that technology was to produce randomized genomic edits, and it does not produce precise, library integrations into the genome. Recently, another group (Mouridi et al, 2022) built on the utility of heatshock Cas9 and integrated three individual sequences from an array. While these prior multiplexed methods made substantial contributions in improving the efficiency of specific transgenesis, none have yet demonstrated multiplexing beyond tens of unique sequences-orders of magnitude below what would be needed for exploratory transgenesis.…”

“…The second component of the TARDIS integration system is a preintegrated landing pad sequence. We have generally favored split selectable landing pads (SSLPs) that use Hygromycin B resistance for its effectiveness (Mouridi et al, 2022;Stevenson et al, 2020Stevenson et al, , 2021. The SSLPs are engineered to accept experiment-specific units from the array.…”

“…Attempts have been made to parallelize transgenic creation in multicellular model organisms, for example, the development of Brainbow (Livet et al, 2007; Weissman and Pan, 2015), ifgMosaic analysis (Pontes-Quero et al, 2017), P[acman] libraries in Drosophila (Venken et al, 2009), and multiple types of transformation in plants (Ismagul et al, 2018; Xu et al, 2022). In Caenorhabditis elegans , CRISPR technology combined with custom engineered sites within the genome (“landing pads”) has facilitated the generation of single-copy integrations (Nonet, 2020; Silva-García et al, 2019; Stevenson et al, 2020), and attempts have been made to multiplex transgenesis using traditional integration methods in conjunction with specialized landing pad systems (Gilleland et al, 2015; Kaymak et al, 2016; Mouridi et al, 2022; Radman et al, 2013). While these efforts have increased throughput over standard single copy integration methods, throughput still remains too low for effective exploratory transgenesis, and in some cases requires significant additional labor, cost, equipment and/or expertise.…”

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS)

“…The second component of the TARDIS integration system is a pre-integrated landing pad sequence. We have generally favored split selectable landing pads (SSLPs) that use Hygromycin B resistance for its effectiveness (Mouridi et al, 2022;Stevenson et al, 2020;. The SSLPs are engineered to accept experiment-specific units from the array.…”