Extended Data Fig. 2: Spatial programming of gene expression.

a. As in Fig. 2b, representative (n = 3) fluorescent microscope image of a photomask stimulation for the PA-TetON (top) and PA-Cre/Lox (bottom) CasRx systems in HEK cells. Magenta: photomask shape. Signal is NG in gray scale. Scale bar: 500 μm. b. As in Fig. 2c, representative (n = 9 single cells from 5 different experiments) images of a single-cell Cre/Lox CasRx stimulation in HEK cells, performed with 100 Hz laser scanning within a confocal microscope setup. Top: confocal image of GFP in grey scale, bottom: transmitted light image. Scale bar: 100 μm. The three FOVs are larger than in Fig. 2c and represent three typical scenarios observed in single-cell photo-stimulations. Left: a single cell is successfully induced; middle: bystander cells are induced as well (moving through the illuminated FOV or deriving by cell division); right: additional cells elsewhere in the FOV are also induced (‘leakage’). c. NG fluorescence quantification for all replicates of single-cell stimulations. A grid of ROIs of ca. the size of a single cell spanning the entire FOV is used for quantifying the signal of the photo-stimulated cell (left ROI, in blue), and of all other ROIs in the FOV (grey), sorted by their distance in micrometers from the photo-stimulated ROI. Signal is normalized (0 to 1) and each replicate is summed with an arbitrary integer to show them stacked (n = 9 FOVs photo-stimulated in 5 experiments). P-value: two-sided t-test. d. Representative (n = 3) live imaging of an organoid treated with 1 μg/ml doxycycline for 24 h and kept in the dark (top) or photo-stimulated with a LED lamp for 24 h (bottom), at 10 days post-stimulation. Magenta: RFP; Green: NeonGreen. Scale bar: 250 μm. e. Live imaging of a time-course as in Fig. 2j for two more organoids locally photo-stimulated via laser scanning (n = 3). Magenta: RFP; Green: NeonGreen. Scale bar: 100 μm.