Fig. 3: Applications of CRISPRoff sgRNAs. | Nature Communications

Fig. 3: Applications of CRISPRoff sgRNAs.

From: CRISPRoff enables spatio-temporal control of CRISPR editing

Fig. 3

a Ratio of on:off-target editing at known off-target sites (OT1 and OT2) for seven sgRNA targets. Ratios were normalized to the maximum on:off ratio as 1 (dark blue) to a minimum of 0.2 (white). Absolute values can be seen in Supplementary Fig. 3. Samples were exposed to light at the indicated time point and harvested after 48 h. DBsgRNAs had a higher on:off-target editing ratio when inactivated at earlier time points. This advantage decreased as editing is allowed to continue. For all targets, standard sgRNAs had the same on:off-target editing regardless of light exposure. b DBsgRNAs are inactivated using LED light from a standard epifluorescent microscope using a 385 nm LED. Full wells were irradiated for the indicated amount of time and analyzed by ICE. Exposure of 6 min ablated editing activity (n = 2 technical replicates, data is presented as mean). c Thin-film mask for spatial patterning using DBsgRNAs. Clear areas are transparent, allowing light to pass through and deactivate DBsgRNAs. Dark areas are opaque and editing continues unimpeded (scale bar: 6.5 mm). d Spatial pattering of gene editing using DBsgRNAs targeting GFP. Cells were irradiated through a negative mask. Editing was terminated in illuminated areas, preserving GFP fluorescence (scale bar: 1 mm). Source data are provided as a Source data file.

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