Figure 1. In vivo labeling of
rrn-4 and
rrn-1 genomic loci using dCas9:NLS::2xEGFP::NLS:(A) Schematic representation for the labeling of genomic loci using the dCas9::NLS::2xEGFP::NLS system. Red stars indicate the amino acid changes in Cas9 to make it nuclease-deficient. (B) Maximum intensity projection of a representative embryo expressing dCas9::NLS::2xEGFP::NLS without sgRNAs. In total, we looked at 7 embryos. The white dotted line marks the eggshell of the embryo. Fluorescence intensity panel: 0% = 0 pixels, 100% = 1925 pixels. (C, left panel) Maximum intensity projection of a representative embryo expressing dCas9::NLS::2xEGFP::NLS with 4 sgRNAs against the
rrn-4 target locus. The white dotted line marks the eggshell of the embryo. The white box highlights a representative nucleus with two foci. Fluorescence intensity panel: 0% = 0 pixels, 100% = 1925 pixels (C, middle panel) The Z-stack (step size = 0.17µm) of the highlighted nucleus with the two foci is shown. (C, right panel) Quantification of the number of foci per nucleus in 6 embryos is shown. The percentage is indicated on top. (D, left panel) Maximum intensity projection of a representative embryo expressing dCas9::NLS::2xEGFP::NLS with 4 sgRNAs against the
rrn-1 target locus. The white dotted line marks the eggshell of the embryo. The white box highlights a representative nucleus with two foci. Fluorescence intensity panel: 0% = 0 pixels, 100% = 1925 pixels (D, middle panel) The Z-stack (step size = 0.17µm) of the highlighted nucleus with the two foci is shown. (D, right panel) Quantification of the number of foci per nucleus in 4 embryos is shown. The percentage is indicated on top. (E, left panel) Maximum intensity projection of a representative embryo expressing dCas9::NLS::2xEGFP::NLS with 4 sgRNAs against each
rrn-4 and
rrn-1 target loci. The white dotted line marks the eggshell of the embryo. The white box highlights a representative nucleus with four foci. Fluorescence intensity panel: 0% = 0 pixels, 100% = 1925 pixels (E, middle panel) The Z-stack (step size = 0.17µm) of the highlighted nucleus with the four foci is shown. (E, right panel) Quantification of the number of foci per nucleus in 6 embryos is shown. The percentage is indicated on top.