Figure 3.
CRISPR Imaging of rDNA Condensation
(A) rDNA condensation upon nutrient depletion or 100 J/m2 UV treatment. dCas9-GFP shows diffuse pattern on uncondensed rDNA but appears as short lines or clusters during condensation. Scale bar, 5 μm (overview) and 1 μm (inset).
(B) Fraction of nuclei showing the condensed rDNA lines and clusters.
(C) Kinetics of rDNA condensation in cells grown in glucose-free liquid culture versus in cells incubated on coverslips. Cells carrying both dCas9-GFP and 9x gRNAs were grown to early log phase in media containing 2% glucose. Glucose was removed at time 0, and cells were imaged at indicated time points. To slow down the condensation process, cells were mounted on a coverslip in a thin layer of media containing 2% glucose (see Transparent Methods for details).
(D) Time-lapse movies of individual yeast cells undergoing rDNA condensation. Cells carrying both dCas9-GFP and 9x gRNAs were mounted on a coverslip in a thin layer of media containing glucose. Widefield images were collected as z stacks, deconvolved, and max projected. For simplicity, only nuclei are shown. Scale bar, 1 μm.
(E) Late condensation process imaged by structured illumination microscopy (SIM). Experiments were set up as in (D), except that cells were incubated on a coverslip for 1 hr to induce rDNA condensation before imaging. Corresponding widefield images are shown (bottom panel). Scale bar, 1 μm.