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. 2019 May 7;9(7):2195–2198. doi: 10.1534/g3.119.400314

Figure 2.

Figure 2

Using the SKI LODGE system to generate single-copy insertions. (A) Flow chart for the generation of single-copy insertions using the SKI LODGE system. (1) Select the SKI LODGE strain to express your gene of interest. Design primers to amplify your gene as homology repair template, including ∼35 bp stretches of SKI LODGE sequences immediately 5′ and 3′ to the dpy-10 site. (2) Assemble CRISPR/Cas9 complex in vitro. (3) Inject pre-assembled CRISPR mix into desired SKI LODGE strain. 3-4 days post injection, isolate individual dumpy/rollers animals, and screen for desired insertion. See step by step guide in Methods section for details. (B) Each SKI LODGE strain was tested by knock-in of a fluorescent protein to confirm tissue-specific expression. wrmScarlet was used to confirm ubiquitous, muscle and neuronal expression from the eft-3, myo-3, and rab-3 SKI LODGE cassettes, respectively. GFP was used to confirm germline expression in the pie-1 SKI LODGE. (c) dpy-10 crRNA::SL2 sequence was introduced into eft-3p::wrmScarlet and rab-3p::wrmScarlet strains to generate alternative SKI LODGE lines. Animals pictured are at day 1 of adulthood.