Figure 4 ∣. In vivo gene activation by hyperdCas12a with single crRNA and poly-crRNA array.

a, Constructs and experimental schematic of in vivo plasmid electroporation in postnatal mouse retina. CAG-GFP is used to mark the electroporated patch. Wildtype CD-1 pups are electroporated on day of birth (P0), and sacrificed at day 21 of life (P21) to access retinal histology. b-d, Representative retinal slices from mouse retina electroporated with a plasmid containing a single crRNA and hyperdCas12a to activate endogenous gene Sox2 (b), Klf4 (c) or Oct4 (d) expression. Note that GFP signal marks the boundary of the electroporated patch, thus the area that did not receive electroporated plasmids serves as an internal control that aids in interpreting the specificity of immunostaining. HA marks the cells that received the plasmid with hyperdCas12a and crRNA. Immunostaining was performed with antibody to Sox2 (panel b), Klf4 (panel c) or Oct4 (panel d) indicating cells that achieved CRISPR endogenous gene activation by single crRNA . e, Representative retina slices of electroporation of a single plasmid containing a poly-crRNA array and hyperdCas12a driving activation of Sox2 (top panel) and Klf4 expression (bottom panel). Box with asterisk (*) marks basal expression of Sox2 in the inner nuclear layer (INL) outside the electroporation boundary. ONL: outer nuclear layer, OPL: outer plexiform layer, IPL: inner plexiform layer, GCL: ganglion cell layer. The images in b-d are representative slices from n=3 independent biological replicates (and are quantitated in Extended Data Fig. 10d). Images in e are representative slides of n=4-5 independent biological replicates (and are quantitated in Fig. 5j-k). Scale bar, 50 μm.