Fig 2: CRISPR-Cas9 Enhances Neuronal Survival in R6/2 Mice(A) Immunofluorescent staining of end-stage striatal sections from R6/2 mice bilaterally injected with 6 × 1010 vector genomes of (left) AAV1-SaCas9-mRosa26 or (right) AAV1-SaCas9-HTT. Insets show high-magnification images. Arrowheads indicate representative DARPP-32+ and SaCas9+ cells with (left) high or (right) reduced mutant HTT (mHTT) protein. Images were captured using identical exposure conditions. Scale bars, 50 µm. (B) Quantitation of immunohistochemical results from R6/2 mice injected with AAV1-SaCas9-HTT (n = 4) or AAV1-SaCas9-mRosa26 (n = 3). *p < 0.05, one-way unpaired t test.

Fig 3: In Vivo Disruption of the Mutant HTT Gene in R6/2 Mice(A) Cartoon of the human HTT transgene located in Gm12695 (predicted gene 12695) on chromosome 4 in R6/2 mice. Arrowhead indicates approximate location of sgRNA binding site. (B) Schematic of the AAV vector. ITR, inverted terminal repeat; hSyn, human synapsin promoter; NLS, nuclear localization sequence; 3xHA, three tandem repeats of the human influenza hemagglutinin (HA) epitope tag. (C) Timeline for in vivo studies. (D) Immunofluorescent staining of striatal sections 4 weeks after R6/2 mice were injected with 6 × 1010 vector genomes of (left) AAV1-SaCas9-mRosa26 or (right) AAV1-SaCas9-HTT. Insets show high-magnification images. Arrowheads indicate representative DARPP-32+ and SaCas9+ cells with (left) high or (right) reduced mutant HTT (mHTT) protein. Images were captured using identical exposure conditions. Scale bars, 50 µm. (E) Quantitation of immunohistochemical results from R6/2 mice injected with AAV1-SaCas9-HTT (n = 3) or AAV1-SaCas9-mRosa26 (n = 3). (F) Normalized mutant HTT protein in striatal lysate 4 weeks after R6/2 mice were injected with 6 × 1010 vg of AAV1-SaCas9-mRosa26 or AAV1-SaCas9-HTT (n = 5). **p < 0.01, unpaired t test. Error bars indicate SD. All injections were performed on 28-day-old animals.

Fig 4: Enrichment of DRD2 neurons and identification of DRD2-expressing interneurons. Fluorescence-based sorting of primary striatal neurons expressing D2 dopamine receptors. (A) Immunocytochemistry of spiny projection neurons (DIV7) expressing DRD2-eGFP, DARPP-32, and DAPI (upper panel) compared to non-sorted primary striatal neurons (lower panel). (B) Identification of DRD2 interneurons which do not express DARPP-32. (C) Quantification of DRD2-eGFP-positive and DARPP-32-negative neurons indicates a 7.9% presence of DRD2 interneurons in sorted cultures (n = 3). (D) Dorsal root ganglionic sensory neurons (DRGs; upper panel) and spinal motor neurons (lower panel) as negative and positive controls for ChAT expression, respectively. (E) Non-sorted striatal cultures (upper panel) and sorted DRD2 neurons (lower panel) showing GFP-positive cholinergic interneurons. Scale bars: 50 µm.