Deletion of Prdm1 and Vsx2 enhancers blocks protein expression. (A) Schematic for the Otx2 gene regulatory network. (B) CRISPR targeting guide design and nomenclature. (C,D,G) Schematic of experimental approaches. Note that electroporations carried out at P0 will not target retinal neurons that are formed embryonically (e.g. cones, horizontals and ganglion cells). (E,H) Immunohistochemistry from electroporated cells. (F) Percentage of electroporated cells that also express PRDM1. (I) Quantification of electroporated cells that also express VSX2. Error bars represent s.d. Significance determined by one-tailed unpaired t-test. ***P<0.001. Notched arrowheads, double-labeled cells; arrowheads, single-labeled cells. Insets show magnification of boxed areas. IHC, immunohistochemistry; N, number of mice used for statistics; ns, not significant; L, lens; ON, optic nerve; R, retina; RPE, retinal pigmented epithelium. Scale bars: 50 µm; 25 µm in insets.