Figure 1. An endogenous nuclear localized SL2:GFP::H2B reporter can be converted to label the cytoplasm by insertion of a T2A sequence:(a) The endogenous locus of
spig-2 (aka
txt-17) was tagged with an SL2::GFP::H2B cassette to label the nuclei of cells expressing this gene. (b) To transform this nuclear-localized reporter into a cytoplasmic one, we used CRISPR/Cas9 genome engineering to cut into the last exon of gfp using the guide RNA shown. (c) Insertion of a STOP codon between GFP and H2B leads to incomplete conversion of the signal from nuclear to cytoplasmic, but the intensity of the GFP is greatly reduced and variable from animal to animal. (d) Insertion of a T2A sequence between GFP and H2B releases GFP into the cytoplasm, labeling the entire cell. The resulting cytoplasmic signal is robust and consistent across animals. The retention of some GFP in the nucleus suggests that the cleavage efficiency of T2A is less than 100%. Scale bars represent 10 μm. The complete sequence of the guide RNA and repair templates can be found in the Reagents section.