Supplementary Figure 2: Confirmation of live-cell imaging and CLING’s specificity. | Nature Structural & Molecular Biology

Supplementary Figure 2: Confirmation of live-cell imaging and CLING’s specificity.

From: Spatiotemporal allele organization by allele-specific CRISPR live-cell imaging (SNP-CLING)

Supplementary Figure 2

(a) Very low laser usage implicates less bleaching and phototoxic effects, ensuring the viability of the cells during time-lapse imaging. Thus, we used a power meter to convert linearly the laser output that is set in percent units in the operating ZEN black software (Zeiss) to µWatt (µW). The grey regions in the plots indicate the bandwidths that were used in all SNP-CLING or CLING experiments. Since a cell has a self-power of ~7 µW, the used laser power was suitable for live cell imaging. The laser at 405 nm was used to image Hoechst 33342 9 (n = 1, Pearson’s correlation). (b) 458 nm were used to visualize GFP fluorescence of the nucleolus (n = 1) and (c) 514 nm for mVenus (n = 1, Pearson’s correlation). (d) mCherry was excited with a 594 nm laser (n = 1, Pearson’s correlation). (e) CLING-signals were characterized in detail to get an impression of the variety of accumulated foci in the nucleus, as well as in the cytosol, in the presence or absence of sgRNAs or dCas9. A sgRNA recognizing telomere sequences was used to establish the transient transfection conditions (n = 10), (Chen, Gilbert et al. 2013). (f) Cytosolic signals in addition to nuclear foci occurred very rarely in some cells, independent of the used sgRNA pool (2/10 nuclei, n = 3, arrowheads = CLING signals of FIRRE and non-specific signals). (g) Transfections without the sgRNA pool generated hazy and cloudy signals throughout the nucleus. Accumulated nuclear punctae were not found (n = 10). (h) In very rare cases one or two foci were observed in a transfection without dCas9 (0.5/10 nuclei, n = 4, arrowheads = CLING signals of CISTR-ACT). (i) An alternating order of MS2-PP7-MS2-PP7 stemloops was cloned into a plasmid to determine whether or not the fluorochromes generated signals that overlap. We used the telomeres sgRNA and determined that the fluorochromes bound specifically. At every foci, both fluorochromes were detected as totally overlapped and mixed signals, as it was previously shown for other fluorochromes and repetitive sequences (Ma, Tu et al. 2016).

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