Fig. 3.

Deletion of the O5 CRM reduced Otx2 transcript levels in bipolar cells. (A,B) The O5-sgRNA5′-1 or -2 plasmid targets the 5′ end of the O5 CRM in the mouse genome, and the O5-sgRNA3′-1 or -2 plasmid targets the 3′ of the O5 CRM. The O5-Cas9 plasmid expressed Cas9 under the control of the O5 CRM. The CAG-mCherry plasmid served as the electroporation efficiency control. These plasmids were co-electroporated into P0 retinas in vivo. At P14, retinas were harvested and dissociated into single cells. The transcript levels of Otx2 in mCherry⁺Chx10(Vsx2)⁺ bipolar cells were detected and quantified by smFISH. (C,D) The level of the Otx2 transcript in control retinal cells. mCherry marked the electroporated cells. Chx10 IHC labeled bipolar cells. The Otx2 transcript was detected by smFISH (green signal). Each green dot in C4 represents one mRNA molecule. D1-D4 are high-magnification views of the highlighted region in C1. White arrows indicate mCherry⁺Chx10⁺ bipolar cells. (E,F) The level of Otx2 transcripts in retinal cells that had the CRISPR constructs to delete the O5 CRM. F1-F4 are high-magnification views of the highlighted region in E1. (G) Quantification of the Otx2 transcript levels. The y-axis represents the number of Otx2 transcripts (green dots) in individual bipolar cells based on smFISH (C4, D3, E4 and F3). Filled circles in the plot represent cells, with each filled circle representing one cell. The percentages of cells that expressed different levels of Otx2 are indicated. sgRNA-pair1: O5-sgRNA5′-1 and O5-sgRNA3′-1; sgRNA-pair2: O5-sgRNA5′-2 and O5-sgRNA3′-2. Student's two-tailed t-test. ****P<0.0001. Scale bars: 10 μm.