Figure 4 | Scientific Reports

Figure 4

From: Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Figure 4

SCN injection with CSAC. (a) Schematic representation of stereotaxic CSAC injection into the SCN. AAVs expressing sgRNAs targeting core clock components and the mCherry fluorescent marker were bilaterally injected into the SCN of Cas9-expressing mice. (b) Representative confocal microscopic image of the brain section containing CSAC-injected SCN. sgRNA-expressing cells are co-labeled with mCherry as a surrogate marker. Scale bar = 100 μm. (c–e) Immunohistochemical analysis of brain sections containing the SCN of mice injected with CSAC-Crys (c), CSAC-Pers (d), and CSAC-Bmal1 (e). Scale bar: 100 μm for (b) and 500 μm for (c–e). (f–h) Co-expression ratio of targeted clock proteins and CSAC. Percentage of clock protein-positive and mCherry-positive cells over mCherry-positive cells were quantified. (f) Co-expression percentages of CRY2 and mCherry are indicated for the sgLacZ or CSAC-Crys group. (g) Co-expression percentages of PER2 and mCherry are indicated for the sgLacZ or CSAC-Pers group. (h) Co-expression percentages of BMAL1 and mCherry are indicated for the sgLacZ or CSAC-Bmal1 group. Error bar = standard deviation (n = 1 mouse for sgLacZ; 3–4 mice for CSAC-Crys, CSAC-Pers, and CSAC-Bmal1, respectively).

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