Picture from Dickinson DJ et al. (2018) MicroPubl Biol "SapTrap assembly of repair templates for Cas9-triggered homologous ...."

Figure 1. A) General organization of SapTrap-assembled constructs. Italics indicate the fragments that are assembled to build the final construct, and blue upper-case letters are the 3-nucleotide overlaps that enable ordered assembly. B) Efficiency of construct assembly (% correct clones) using SapTrap (Schwartz and Jorgensen 2016) or a ccdB/Gibson-based approach (Dickinson et al. 2015).

Picture from Roy, Vincent et al. MicroPubl Biol

Figure 1. mNG::PAR-4A & C are ubiquitously expressed, excluded from nuclei and enriched at the cell cortex. (A) Schematic depiction of par-4 locus organization and the predicted transcripts from this gene before editing (top), after homologous recombination (middle), and after SEC removal (bottom). Adapted from (Dickinson et al. 2015). (B-E) Fluorescence micrographs (B, D) and pseudo-DIC/fluorescence-merge micrographs (C, E) of wild-type (N2) animals and mNG::par-4a & c animals taken with the same settings, 24 hours (25 degrees C) after the late L4 stage. (F) Fluorescence micrograph showing a close-up of the germ line of a mNG::par-4a & c animal. (G) Fluorescence micrographs of 1- and 4-cell mNG::par-4a & c embryos. Scale bars: B-E, 80 mM; F, 55 mM; G, 27 mM. (H) Maternal effect embryonic lethality of the indicated genotypes at 25 degrees C.

Picture from Witten G et al. (2023) MicroPubl Biol "mScarlet and split fluorophore mScarlet resources for plasmid-based ...."

Figure 1. Analysis of HIS-72, EGL-1, and PTL-1 split wrmScarlet tags in C. elegans:(A) Plasmid maps for the mScarlet knock-in constructs developed as part of this project. pGLOW39 and pGLOW63 allow tagging of any gene of interest (GOI) with mScarlet-I-C1 or split fluorophore wrmScarlet11, respectively. ccdB regions are removed by DNA digest and homology arms for the GOI are cloned as described by Dickinson et al. 2015. pGLOW119 allows cloning of any promoter upstream of split fluorophore wrmScarlet1-10, and this construct can be inserted via CRISPR/Cas9 homologous recombination into the ttTi5605 locus on LGII. (B) Testing the split fluorophore system using HIS-72. (Top) Endogenous expression of tiny-tagged HIS-72 (his-72::3xMyc::wrmScarlet11 in an muIs252 (Peft-3::wrmScarlet1-10) genetic background. HIS-72 is visible in all somatic nuclei, consistent with Goudeau et al. 2021. This confirms that the 3xMyc epitope tag does not interfere with complementation of the split fluorophore. (Middle) We created an alternative somatic expression construct for wrmScarlet1-10 using the his-72 promoter and tbb-2 (3'UTR) rather than the eft-3 promoter and unc-54 (3'UTR). Both somatic wrmScarlet1-10 constructs appear to have ubiquitous expression in all nuclei (compare top and middle panels). (Bottom) We also created a construct for neuronal expression of wrmScarlet1-10 using the rab-3 promoter. Note that HIS-72::wrmScarlet11 and wrmScarlet1-10 complementation is visible in the neuronal nuclei of the head and ventral nerve cord. (C) egl-1 functions within the V5.paa lineages to eliminate the sister cell of PVD via programmed cell death. (D) The EGL-1 tiny tag is functional. We confirmed that the PVD sister cells undergo apoptosis in animals expressing egl-1::wrmScarlet11 and Peft-3::wrmScarlet1-10. The dopaminergic neuron marker Pdat-1::gfp was used to identify PDE neurons and "undead" PVD sister cells in L3 worms. The relevant cell bodies are circled in white. (E) Animals with wild type EGL-1 or tiny-tagged EGL-1 have just 2 PDE neurons, whereas egl-1 knockout worms have an average of 3 (but sometimes 4) PDE and PDE-like neurons (p<0.0001, unpaired t-test). (F) To observe EGL-1 tiny tag expression, we induced programmed cell death in germ cells using UV irradiation. At 6 hours post-UV, we were unable to observe any expression in the germline (mitotic region nor death zone). Yellow arrowheads indicate out-of-focus, round, autofluorescent gut granules (i.e. halo effect). (G) N-terminal tiny-tagging strategy for the ptl-1 gene. Isoforms A-C share a common first exon, so we opted to do a 5' knock-in of wrmScarlet11 at this exon to simultaneously tag multiple isoforms of PTL-1. (H) Mechanosensation indicates the PTL-1 tiny tag is functional. Animals bearing tiny-tagged PTL-1(N) responded to gentle touch just as well as animals bearing wild-type PTL-1 (p>0.05, paired t-tests). (I) PTL-1 is expressed in the axons of posterior mechanosensory neurons PVM, PLMR, and PLML. (Top) DIC image of the tail of a young adult worm, with the structures of PVM, PLMR, and PLML superimposed diagrammatically in black. Circles represent the cell bodies, whereas lines represent the axons. (Bottom) We were unable to observe any PTL-1(N) expression in the posterior mechanosensory neurons of wrmScarlet11::ptl-1(N); Prab-3::wrmScarlet1-10 animals using epifluorescence microscopy at 40x, the highest resolution available in our laboratory.