Fig. 2 |. Tethering of RNA transcripts to DSB recapitulates the effects of local transcription on HR.
a, Schematic of dCas9-mediated tethering of fusion RNAs to the vicinity of DSB. The fusion RNAg-t-GFP containing guide RNA (red), tracr RNA (blue) and a 120-nt GFP sequence that flanks the I-SceI site (green) is transcribed from a U6 promoter on a plasmid. RNAg-t-GFP is targeted by dCas9 to a unique sequence 5′ to the I-SceI site. b, The indicated fusion RNAs were targeted as in a in the transcriptionally off state (−Dox). The HR efficiencies of cells expressing fusion RNAs were determined by qPCR and normalized to the HR efficiency of cells expressing an inert RNA (tracr RNA) in the transcriptionally on state (+Dox). Data are mean ± s.e.m. (n = 3 independent experiments). c, d, Fusion RNAs containing sense GFP, anti-sense GFP or scrambled sequences were targeted 5′ (c) or 3′ (d) to the I-SceI site. HR efficiency was measured by qPCR in transcriptionally on and off states (+Dox and −Dox, respectively). Ratios of HR efficiencies between the off and on states (−Dox/+Dox) were determined. Data are mean ± s.e.m. (n = 3 independent experiments).