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. Author manuscript; available in PMC: 2024 Aug 21.
Published in final edited form as: Cell Rep. 2024 Jul 1;43(7):114420. doi: 10.1016/j.celrep.2024.114420

Figure 1. DNA double-strand break induced by vfCRISPR causes rapid transcription repression that coincides with 53BP1 recruitment.

Figure 1.

(A) Schematic of live-cell reporter to simultaneously monitor transcription and DNA damage repair. The reporter was under the control of a Tet-On promoter and encoded cyan fluorescent protein (CFP). 24xMBS was inserted in the 3′ UTR. MCP-GFP bound to the MBS and labeled the transcription site (TS). A caged guide RNA (cgRNA) was designed to target a site downstream of the MBS. When stimulated by light, Cas9/cgRNA was activated and created a DSB. mCherry-53BP1 stably expressed in the cell was recruited to the DSB to mark the damaged DNA.

(B) TS of the live-cell reporter was identified before stimulation. Green: MCP-GFP; red: mCherry-53BP1; white arrowhead: TS. Scale bar: 5 μm.

(C) DSB was created by scanning a diffraction-limited 405 nm laser beam surrounding the TS. The transcription and DSB repair were monitored by time-lapse imaging (Video S1). Snapshots showed the TS’s disappearance and 53BP1’s recruitment to the damaged locus. Scale bar: 1 μm.

(D) Fluorescence intensity traces of TS and 53BP1 in (C). Intensities were normalized to the maximum value of each trace.

(E) Heatmaps of TS and 53BP1 intensities. Top: cells with 53BP1 recruitment within 30 min, n = 170 out of 267 total stimulated cells. Bottom: the exact procedure was performed on cells electroporated with a mutant guide RNA that bound to but did not cut the DNA target, n = 68 cells. Each row represents an individual cell, and the color denotes normalized fluorescence intensity. Cells are ranked by the sum of normalized TS intensity.

(F) The averaged TS (top) and 53BP1 (bottom) intensities as shown in (E). The negative control is shown in gray. Shading represents the 95% confidence interval.

(G) Top: fitting of TS intensity trace with a delayed exponential decay model. τ, decay time; δ, response time. Bottom: a representative TS intensity trace (symbol) and the fitting to the model (curve).

(H) Scatterplots of decay (left) and response (right) times from fitting TS traces in (E). Lines: quartiles.