Extended Data Fig. 1: Development of a live-cell Y chromosome-labelling system by targeting dCas9-SunTag to the DYZ1 array.
From: Mitotic clustering of pulverized chromosomes from micronuclei
a) Images of DLD-1 cell populations expressing dCas9-SunTag and sfGFP-scFv with the indicated sgRNAs targeting the DYZ1 array. Scale bar, 5 μm. b) List of sgRNA sequences used in (a). sgDYZ1-2 was used for the remainder of the study. c) Images of DLD-1 cell populations expressing sfGFP-scFv under the control of full-length or truncated CMV promoters with dCas9-SunTag containing the indicated scaffold lengths. Scale bar, 5 μm. d) Signal-to-noise measurements for the conditions shown in (c). Data represent mean; from left to right, n = 13, 13, 15, 11, 12, and 10 cells. e) IF-FISH image of interphase cells showing co-localization between an anti-GFP antibody recognizing sfGFP bound to dCas9-SunTag and DNA FISH probes targeting the Y chromosome q-arm heterochromatic array (YqH). Scale bar, 5 μm. f) Fluorescent line scan analysis of the indicated region marked in (e) showing high specificity of the SunTag with YqH FISH. g) IF-FISH image of mitotic chromosomes showing co-localization between an anti-GFP antibody recognizing dCas9-SunTag with chromosome paint probes targeting YqH. Scale bar, 10 μm. h) Example images of live DLD-1 cells with dCas9-SunTag signals in the nucleus or micronucleus. Scale bar, 5 μm. i) Proportion of nuclei and micronuclei with or without dCas9-SunTag signals following DOX/IAA induction for the indicated number of days. Data represent mean ± SEM of n = 3 independent experiments; 0 days = 1,044, 2 days = 1,070, 3 days = 1,123 cells. j) Background fluorescence measurements of non-dCas9-SunTag-bound sfGFP-scFv from n = 13 micronuclei obtained from independent experiments (left) and schematic of intact and ruptured micronuclei measurements (right).
