Fig. 4

Descending LC-NAergic neuron-specific NA transporter knockout by CRISPR-Cas9 ameliorates chronic itch. a A schematic illustration of retrograde transduction strategy of SaCas9 and guide RNA [sgSlc6a2 for NA transporter (NET) and sgRosa (as control)] expression in descending LC-NAergic neurons. b Representative images of SaCs9 (detected by HA immunostaining, green) and TH (red) expression in LC-NAergic neurons. Scale bar, 200 μm. c Downregulation of NET (green) in descending LC-NAergic neurons (visualized by mCherry (red) and TH immunostaining (cyan)) in sgSlc6a2-expressing mice. Scale bar, 200 μm. Quantitative analysis of the immunofluorescence intensity of NET (d: n = 4 mice, ***P < 0.001) and TH (e: n = 4 mice). Effect of genome editing of NET on DCP-induced scratching behavior (f, *P < 0.05 vs. sgRosa-group on day 14), dermatitis [g (representative photomicrographs) and h, **P < 0.01 vs. sgRosa-group on day 14) and TEWL (i, **P < 0.01 vs. sgRosa-group on day 14) in DCP-treated mice (sgRosa, n = 8; sgSlc6a2, n = 10). Data show the mean ± SEM