Fig 1: Tagging endogenous arrestin with EGFP in the postnatal mouse retina. (A) Structures of the mouse arrestin locus and the knock-in targeting vector to produce an arrestin-EGFP fusion protein. The predicted Cas9-gRNA cutting position is indicated with a scissor symbol. (B) Mouse retinas were co-electroporated at P0 with Arrestin targeting vector (EGFP), CAG-H2B-mCherry, and CBh-Cas9 with (+) or without (-) gRNA for arrestin. Fluorescent images of intact retinas harvested at P21 are shown. CRISPR/Cas9-dependent gene knock-in at the arrestin locus occurred at a frequency of ~1% in the electroporated retinas. White dashed rectangles indicate the regions that are presented at higher magnification in the rightmost panels. (C) Retinas were collected from the light-adapted (left panels) or dark-adapted mice (right panels), sectioned, and stained with anti-GFP (green) and anti-RFP (red) antibodies and DAPI (blue). EGFP signals were detected only in rod photoreceptors. In the light-adapted retinas, EGFP signals were predominantly localized in the outer segments of rod photoreceptors, while in the dark-adapted retinas, EGFP signals were localized in the inner segments and cell bodies but not in the outer segments of rod photoreceptors. OS, outer segment; IS, inner segment; ONL, outer nuclear layer.