Figure 3.

CRISPR/Cas9 gene editing of Hsp90α in adult mouse brain. CRISPR constructs for all targets prepared and delivered as described in “Materials and Methods” section. All quantitative data reported as mean ± SEM. (A) Hsp90α CRISPR construct validated in 66.1 cells. Western blot shown with replicate wells of cells in each lane, with MW indicated for each target. Hsp90α protein levels reduced by ~90% only in the presence of Hsp90α-targeted CRISPR construct. Other constructs (Negative Control, PEBP) have no effect. mCherry protein levels are present in all transfected cells, demonstrating successful transfection of CRISPR DNA. (B) Hsp90α or negative control CRISPR delivered to CD-1 male mouse brains and analyzed for protein knockdown on day 10. Representative images shown from pontine reticular nucleus (PRN) and periaqueductal gray (PAG). Hsp90α (green signal) is present in cell bodies and dendritic trees, and the signal is reduced by CRISPR treatment. (C) Quantitation of all data from (B) performed as described in “Materials and Methods” section. Sample size of mice/group noted in graph. **p < 0.01 vs. Negative Control group by Unpaired 2-Tailed t-test. Mice treated in one technical replicate, with the resulting tissue stained and analyzed in more than one technical replicate. CRISPR treatment reduced Hsp90α signal by 43.9%. (D) CRISPR-treated CD-1 male mice had paw incision surgery performed on day 9, with injection of 3.2 mg/kg morphine sc on day 10. Sample size of mice/group noted in graph, performed in two technical replicates. *,**,***p < 0.05, 0.01, 0.001 vs. same time point Hsp90α group by two-way ANOVA with Fisher’s Least Significant Difference post hoc test.