Figure 2.

Allele-specific targeting and disruption of Rho^S334 in vivo. (a) Fast-Green DNA dye shows plasmid distribution following unilateral subretinal injection in S334ter-3 rats at P0. (b) Representative retinal flat-mount shows variable mCherry intensity and uneven distribution 4 days after injection. (c) Gating strategy for fluorescence-activated cell sorting isolation of enzymatically dissociated P4 retinal cells with mCherry fluorescence intensity at high (red, 0.3%) intermediate (purple, 0.6%), and no (blue, 97.9%) expression. Sanger sequencing of PCR-amplified genomic Rho loci from photoreceptors showed in vivo disruption of Rho^S334 (d), but not Rho^WT (e) using gRNA^TRGT. (f) Rho^S334 locus targeting schematic and deep sequencing reads shows insertions/deletions (indels) proximal to the predicted cleavage site (arrowhead) from gRNA^TRGT-expressing cells. PAM, protospacer adjacent motif.