Supplementary Figure 6: Analysis of innate immune response in sham treated, RNP treated and untreated brains.

A) Morphological appearance of microglia in the mouse striatum in Cas9 RNP complex RNP treated and untreated mice visualized in green by immunostaining with anti-Iba-1 antibody. Iba-1 (ionized calcium-binding adapter molecule 1, also known as Allograft inflammatory factor 1 (AIF-1)) is useful as an indicator of activated microglia because 1) its levels increase and 2) the cytoplasmic staining pattern can be used to assess microglia morphological changes that occur upon activation, i.e. microglia enlarge their cell bodies and thicken their processes, which closely enwrap neuronal cell bodies²⁹. In Cas9 RNP treated mice, microglia have small cell bodies and long and slender processes indicating they are not activated. B) Quantification of Iba-1 protein intensity in untreated, sham (buffer only) and 4x-NLS-Cas9-2xNLS treated mice. Data are represented as mean ± SEM with scatter plots of mean intensity of 2-8 sections per animal (One way ANOVA; p = 0.4496 and F_{2, 7} = 0.898). n=3 animals per condition with 2-8 sections analyzed per animal. For each section, fluorescence intensity of the Iba-1 labeled channel for the entire 20X field was measured with image J. The experimenter was blinded to treatment condition while performing quantitation of fluorescence intensity. C) A panel of microglia markers of activation were analyzed at two timepoints, 3 and 12 days post injection by qPCR transcript analysis. The panel contains: CD11b, CD45, CD68, CD86, Cx3cr1, IBA-1, IL-12b p40, IL-12a p35, Tmem119 and TNF-α. Dorsal striatum of sham injected (buffer only) and Cas9 RNP (50pmol/0.5ul) injected mice were collected at 3 days post injection (n=3 animals, n=2 bilateral injections per animal) and 12 days post injection (n=3 animals, n=2 bilateral injections per animal). Transcripts that are significantly different in sham vs. RNP injected at 3 days post injection are Cd11b, Cx3cr1, IL-12b p40 and IL-12a p35. All are depleted in RNP-treated samples relative to sham-treated. At 12-day post injection, TNF-α is the only transcript significantly different and increased 5.65-fold in RNP-treated relative to sham-treated samples. Data are presented as fold change relative to sham for respective timepoint. Data are represented as mean ± SEM with scatter plots of individual samples. Data for 3 days post injection. Two-tailed unpaired t test with equal SD was used to assess significance for the following genes: Cd11b p=0.0378 and F_5,5 2.903, CD45 p=0.1298 and F_5,5 2.133, CD68 p=0.0718 and F_5,5 32.36, CD86 p=0.3150 and F_5,5 1.22, Cx3cr1 p=0.0371 and F_5,5 12239, IBA-1 p=0.1100 and F_5,5 6.714, IL-12b p40 p=0.0322 and F_5,5 3.71, IL-12a p35 p=0.0138 and F_5,5 4.44, Tmem119 p=0.1946 and F_5,5 1.518, TNF-α p=0.9248 and F_5,5 2.776. Data for 12 days post injection. Two-tailed unpaired t test with equal SD was used to assess significance for the following genes: Cd11b p=0.1072 and F_5,5 7.168, CD45 p=0.4906 and F_5,5 2.177, CD68 p=0.0562 and F_5,5 10.72, Cx3cr1 p=0.1548 and F_5,5 217, IBA-1 p=0.3129 and F_5,5 3.355, IL-12b p40 p=0.0625 and F_5,5 4.272, IL-12a p35 p=0.0657 and F_5,5 14.54, P2ry12 p=0.0760 and F_5,5 67.41, TNF-α p=0.0384 and F_5,5 23.81.