CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington’s Disease Mouse Model

Figures

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  Figure 1. Disruption of the Mutant HTT Gene in Reporter Cells Using CRISPR-Cas9 Nucleases (A) Schematic representation of the human HTT locus located on chromosome 4 and the candidate sgRNA target sites. Arrowheads indicate the approximate location of the sgRNA binding site. aa, amino acids; PAM, protospacer-adjacent motif. (B) Top: graphic of the mutant HTT-CFP reporter. Bottom: the percentage of CFP-positive HEK293T cells 72 h after transfection with reporter plasmid, SaCas9, and the HTT-targeting sgRNAs or a non-targeted sgRNA (Empty) (n = 3). (C) Top: western blot of lysate from HEK293T cells 72 h after transfection with reporter plasmid, SaCas9, and the HTT-targeting sgRNAs or a non-targeted sgRNA (Empty). Bottom: quantitation of western blot results. CFP protein was normalized to GAPDH protein in each lane (n = 3). Error bars indicate SD. **p < 0.01; ***p < 0.001, unpaired t test.

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  Figure 2. 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 <…

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  Figure 3. CRISPR-Cas9-Mediated Disruption of the Mutant HTT Gene Provides Therapeutic Benefit to R6/2 Mice (A–D) Mean survival (A), percent survival (B), normalized rotarod (C), and normalized clasping (D) scores for R6/2 mice bilaterally injected with 6 × 1010 vg of AAV1-SaCas9-HTT (n = 10) and AAV1-SaCas9-mRosa26 (n = 10). Wild-type mice (n = 11) are litter-matching B6CBAF1 mice. Values are means, and error bars indicate SEM. *p < 0.05. In (B), unpaired t test was used; in (C) and (D), a two-way ANOVA was used, followed by Tukey’s post hoc analysis.

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  Figure 4. 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.