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. 2022 Aug 25;12(9):jkac184.
doi: 10.1093/g3journal/jkac184.

Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans

Sonia El Mouridi  1 , Faisal Alkhaldi  1 , Christian Frøkjær-Jensen  1
Affiliations

Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans

Sonia El Mouridi et al. G3 (Bethesda). .

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 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.

Keywords: Caenorhabditis elegans; CRISPR/Cas9; genome engineering; long-read assembly; transgenesis.

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Figures

Fig. 1.
Fig. 1.
Schematic of MosTI protocol. a) (1) Target transgenes are generated by cloning (standard restriction-enzyme, Gateway, Golden Gate, or gene synthesis) into a MosTI-compatible plasmid containing a nonrescuing cbr-unc-119 rescue fragment. (2) Target plasmids are coinjected with Cas9 and sgRNA plasmids into unc-119(ed3) animals containing a MosTI landing site. Antibiotic or fluorescent markers can be used to enrich the population for nonrescued transgenic array animals (optional). (3) Cas9-induced DSBs are repaired by homology-directed repair from the target plasmid, leading to transgene insertion and reconstitution of a functional cbr-unc-119(+) gene. (4) Animals with single-copy insertions are identified by phenotypic rescue (N2-like animals on plates with Unc animals). (5) The extrachromosomal array is lost, and the cbr-unc-119 selection can be removed by Cre expression (optional). b) MosTI-compatible insertion sites in safe-harbor landing sites were selected for permissive chromatin environment (Ho et al. 2014). c) A collection of MosTI-compatible cloning vectors for tissue-specific gene expression, transcriptional and translational fluorescence expression, and fluorescence coexpression with a gpd-2 operon has been deposited with Addgene (see Supplementary Fig. 3 for all plasmids).
Fig. 2.
Fig. 2.
MosTI insertion frequency and transgene expression. a) Table with MosTI insertion efficiencies using constitutive (Psmu-2::Cas9) or heat-shock inducible (Phsp-16.41::Cas9) plasmids for Cas9 expression. b) Single-copy transgenes (Peft-3::gfp) are widely expressed, including the germline (13/13 inserts expressed). Left panels ×10 magnification, right panels ×40 magnification. Scale bars: 20 µm. c) GFP quantification by wide-field fluorescence microscopy (L4 stage) from single-copy Peft-3::gfp insertions at three MosTI landing sites. Statistics: Kruskal–Wallis’s test of fluorescence intensity grouped by insertion site (chr. I: 35 images, n = 2; chr. II: 41 images, n = 3; chr. IV: 13 images, n = 1 insert, *P < 0.05).
Fig. 3.
Fig. 3.
Single-copy insertions from multiplex transgene injections. a) Schematic of multiplex insertions using MosTI. Multiple transgenes are pooled in one injection mix. During homology-directed repair, a single transgene from the array is inserted into the MosTI site. b) Quantification of single-copy MosTI insertions over seven generations from four independent transgenic animals with extrachromosomal arrays containing three target transgenes (Prpl-7A::gfp, Peft-3::mMaple3, and Peft-3::mScarlet) and a heat-shock inducible Cas9 (Phsp-16.21::Cas9). In each generation, a new population of animals was generated from a nonrescued (Unc) animal carrying an extrachromosomal array and these animals were heat-shocked 1 or 2 days before exhausting the bacterial lawn. c) Fluorescence microscopy showing expression from single-copy insertions of each of the 3 target transgenes. ×20 magnification, scale bar = 20 µm.
Fig. 4.
Fig. 4.
MosTI sites are modular and can be converted to other split selection markers. a) Schematic of the conversion of an unc-119 MosTI site into a MosTI site that uses a different split selection marker (here, HygroR or Pmlc-2::gfp). (1) Injection of a conversion plasmid (e.g. generated by gene synthesis) into unc-119(ed3) mutants with a MosTI landing site. (2) Insertion of the conversion plasmid generates a new MosTI landing site using a different split selection marker. (3) Loss of the extrachromosomal array and (optional) excision of the reconstituted cbr-unc-119 selection marker. (4) Injection of a modified MosTI target vector uses the novel selection marker to insert a single-copy transgene. b) Available MosTI sites for split Pmlc-2::gfp-nls and HygroR selection. c) Example of a single-copy insertion of a Pmex-5::gfp (germline) transgene into a converted MosTI site using a split pan-muscular gfp selection marker (Pmlc-2::gfp-nls). Top panel: pan-muscular nuclear GFP expression (L2 stage). Bottom panel: nuclear GFP expression in muscles (from pan-muscular selection) and in the germline (from the Pmex-5::gfp-nls) transgene. Top panel ×20 magnification, bottom panel ×40 magnification. Scale bar: 20 µm.
Fig. 5.
Fig. 5.
Targeted integration of extrachromosomal arrays at MosTI sites and at the endogenous ce-unc-119 locus. a) Schematic of “two-step plasmid” integration of extrachromosomal arrays at a MosTI landing site. (1) An extrachromosomal array is formed by injecting transgenes, including a plasmid containing the fourth intron of cbr-unc-119 (integration fragment) into unc-119(ed3) animals containing a MosTI landing site. (2) Plasmids expressing Cas9 and an sgRNA injected into transgenic animals cause DSBs at the MosTI landing site and in the array (in integration fragments). (3) DSBs are repaired by NHEJ between the array and the MosTI site, resulting in unc-119 rescue. The sgRNA target is in an intron to allow rescue even when NHEJ repair causes short indels. Note, arrays can also be inserted by a similar strategy at the ce-unc-119(ed3) locus, or by injecting Cas9 protein and crRNA/tracrRNA. b) Fluorescence microscopy of an integrated extrachromosomal array containing transgenes expressed in muscles (Pmlc-1::gfp and Prab-3::mCherry) inserted into a MosTI site on chr. II. Scale bar = 20 µm. c) Fluorescence microscopy of an integrated array (Pmlc-1::gfp and Pmlc-1::tagRFP) inserted into the endogenous ce-unc-119(ed3) locus (chr. III). Scale bar = 20 µm. d) Table showing the efficiency of extrachromosomal array insertion using 1- and 2-step protocols at MosTI landing sites and at ce-unc-119(ed3).
Fig. 6.
Fig. 6.
Molecular characterization of targeted array integrations. a) Analysis of arrays integrated at the ce-unc-119 locus using a “1-step plasmid” protocol (left) and a “2-step protein” protocol (right). Top: fluorescence images of strains with array integrations. Scale bar = 20 µm. Middle: quantification of total fluorescence in adult animals using a COPAS large-particle fluorescence sorter. Bottom: plasmid copy-number estimates based on WGS (Illumina short read sequencing). b) Integrated array assembly from long-read Oxford Nanopore sequencing of CFJ157 (2-step protein array integration containing gfp, tagRFP, hygroR, integration fragment, and 1 kb plus ladder). Top: schematic overview of 4 long contigs. Bottom: individual areas from contigs (location indicated by numbers above). Arrows indicate the transcriptional direction of transgenes (promoter::gene::UTR). c) Assembly statistics from the strain CFJ157. d) Comparison of copy-number estimates based on Illumina short-read sequencing and Oxford Nanopore long-read sequencing of CFJ157.
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