Multiplexed CRISPR/Cas9 Targeting of Genes Implicated in Retinal Regeneration and Degeneration

Unal Eroglu, A., Mulligan, T.S., Zhang, L., White, D.T., Sengupta, S., Nie, C., Lu, N.Y., Qian, J., Xu, L., Pei, W., Burgess, S.M., Saxena, M.T., Mumm, J.S.
Full text @ Front Cell Dev Biol

Genotype and phenotype of the CRISPR/Cas9-induced rhodjh503 mutation. (A) Sequence of the wildtype rho coding sequence targeted by CRISPR/cas9 (sgRNA sequence underlined) and the location of the 5 bp deletion in the rhodjh503 mutant allele. The amino acid sequences predicted to result from these open reading frames are shown below the nucleotide sequence with the altered sequence highlighted in red. (B) YFP signal intensity changes in rho+/+ fish and rhodjh503+/− mutant fish from 3 to 7 dpf (±sd). Daily fluorescence microplate readings of rho+/+ fish and rhodjh503+/− mutants in the rho:YFP-NTR background. The roy control group have no YFP-expressing transgene. Pairwise comparisons (i.e., T-test followed by Bonferroni correction for multiple comparisons) between per day data points produced p-values of ≤ 0.0005. (C) Composite maximum intensity projection images of confocal Z-stacks taken of the eyes of wildtype and mutant fish from 3 to 7 dpf. 3, 4, and 5 dpf images were taken with a 2x zoom while the 6–7 dpf images were taken with a 1.8x zoom. Several of the photoreceptors present in the mutant retinas are indicated by red arrows. The scale bar represents 50 μm in the 3–5 dpf images and 55.6 μm in the 6 and 7 dpf images. AA, amino acid; bp, base pair; cds, coding sequence; dpf, days post-fertilization; WT, wildtype; YFP, yellow fluorescent protein.

Rod but not cone photoreceptors are reduced in rhodjh503+/− mutants. (A) The 1d1 antibody (magenta), which recognizes Rhodopsin, labels the outer segments of wildtype rod cells. The labeling pattern is similar in both wildtype and mutants at 3 dpf. At 6 dpf, 1d1 antibody labeling is restricted to the proliferating marginal zone in rhodjh503+/− mutants (*), but remains throughout the ONL in wildtype retinas. The white boxed region in the 6 dpf images are enlarged in the panels to the right showing that the 1d1 antibody labels YFP-expressing rod cells (green, arrows). (B) The zpr1 (magenta) antibody labeled cone cells do not show an obvious difference between wildtype and mutant retinas at 4 and 7 dpf. The boxed region in the 7 dpf images is enlarged to the right showing no overlay between zpr1 antibody staining and YFP-expressing rod cells (green). DAPI (blue) was used to stain cell nuclei. Arr3a, Arrestin 3a; DAPI, 4′,6-diamidino-2-phenylindole; dpf, days post-fertilization; ONL, outer nuclear layer; rho, rhodopsin; Rho, Rhodopsin; YFP, yellow fluorescent protein.

The number of apoptotic cells in the ONL is increased in rho djh503c+/− mutant retinas at early developmental stages. (A) Representative images of TUNEL stained retinas from 3 to 5 dpf. In wildtype retinas, TUNEL positive cells (magenta) were rarely seen in the ONL from 3 to 5 dpf. In contrast, TUNEL positive cells were frequently detected in rhodjh503+/− mutants at 3 and 4 dpf, but only occasionally at 5 dpf. Rod cells are labeled by YFP (green). (B) Quantification of TUNEL positive cells in the ONL. The number of TUNEL positive cells was significantly higher in rhodjh503+/−mutants than in wildtype at 3 and 4 dpf, but not 5 dpf. Mann-Whitney test p-values: **p < 0.001, *p < 0.01. Sample size (n) of each condition is provided. DAPI (blue) was used to stain nuclei. dpf, days post-fertilization; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; rho, rhodopsin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling.

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Front Cell Dev Biol