S10 peptide delivery of Cas9 RNP shows editing in ROSAmT/mG locus in vivo. Cas9 RNP directed to loxP sites flanking the tdTomato cassette were administered with S10 peptide once daily on 2 consecutive days. Seven days later, conversion of tdTomato to GFP expression was visualized in lung tissue sections. A) Schematic of gRNA targeting two loxP sequences flanking tdTomato gene and experimental protocol for Cas9 RNP delivery to ROSAmT/mG mice. B) Fluorescence image of large airway 7 days following two intranasal doses of [S10]: 40 µM; [Cas9]: 1.33 µM; [gRNA]: 2 µM; ×2 magnification. GFP expression denotes edited cells. C) Editing in a large airway, ×20 magnification. D) Editing in a small airway; ×20 magnification. E) Co-localization of GFP and marker of ciliated cells (α-tubulin, white) in large airway. Arrows indicate α-tubulin co-localization with GFP. Arrowheads denote edited (GFP+) nonciliated cells negative for α-tubulin; ×40 magnification. F) Co-localization of GFP+ and SP-C (white) identifies alveolar type II cells (arrows) in distal lung; ×40 magnification. G) Representative image of large airway after delivery of Cas9 RNP alone shows no editing, ×20 magnification. H) Representative image of small airways after delivery of Cas9 RNP alone shows no editing; ×20 magnification. I) Editing efficiency of Cas9 in large and small airways quantified by the number of GFP+ cells. Horizontal lines indicate mean ± SE; n = 5 mice/group. Pubmed