Shortening the Half-Life of Cas9 Maintains Its Gene Editing Ability and Reduces Neuronal Toxicity

Abstract

Virus-mediated expression of CRISPR/Cas9 is commonly used for genome editing in animal brains to model or treat neurological diseases, but the potential neurotoxicity of overexpressing bacterial Cas9 in the mammalian brain remains unknown. Through RNA sequencing (RNA-seq) analysis, we find that virus-mediated expression of Cas9 influences the expression of genes involved in neuronal functions. Reducing the half-life of Cas9 by tagging with geminin, whose expression is regulated by the cell cycle, maintains the genome editing capacity of Cas9 but significantly alleviates neurotoxicity. Thus, modification of Cas9 by shortening its half-life can help develop CRISPR/Cas9-based therapeutic approaches for treating neurological disorders.

Keywords: CRISPR/Cas9; genome editing; neurotoxicity.

Figure 1.. Validation of AAV-GCas9 In Vitro

(A) Schematic representation of the expression of Geminin during cell-cycle progression. Geminin is expressed in the cycle phases indicated with red line and is degraded in the quiescent phase (G1/G0). (B) Schematic representation of AAV-Cas9 and AAV-GCas9 constructs. (C) The expression of Cas9 and GCas9 was examined in nocodazole-treated N2a cells at 0, 1, 2, 4, 6, and 8 hr after removing nocodazole. Actin was used as a loading control. Cyclin D1 and Geminin expression were used to indicate the progression of cell cycle from M to G1 phase. (D) Quantitative analysis of western blotting results in (C) (n = 3; **p < 0.01; two-way ANOVA with Bonferroni post-tests). *p < 0.05. (E) The expression of Cas9 and GCas9 was examined in N2a cells at 0, 1, 2, 4, 6, and 8 hr after cycloheximide treatment. Actin was used as a loading control. (F) Quantitative analysis of western blotting results in (E) (n = 3; **p < 0.01; ***p < 0.001; two-way ANOVA with Bonferroni post-tests). Data are represented as mean ± SEM.

Figure 2.. Validation of AAV-GCas9 In Vivo

(A) Immunohistochemistry showing the expression of Cas9 protein using hemagglutinin (HA) antibody in the striatum of wild-type (WT) mice 2 weeks after injection of AAV-Cas9 or AAV-GCas9 (scale bar: 50 μm). (B) Quantitative analysis of immunohistochemistry results in (A) (n = 5; ****p < 0.0001; one-way ANOVA with Tukey’s post-tests). (C) Western blot examination of the expression of Cas9 and Manf in the striatum of 3-month-old WT mice injected with AAV-Cas9, AAV-GCas9, AAV-Cas9 diluted 10 times, or AAV-Cas9 diluted 100 times. Actin was used as a loading control. (D) Quantitative analysis of western blotting results in (C) (n = 5; *p < 0.05; ***p < 0.001; ****p < 0.0001; one-way ANOVA with Tukey’s post-tests). (E) Immunohistochemistry showed the removal of mutant Huntingtin in the striatum of heterozygous 3-month-old HD140Q KI mice injected with AAV-Cas9 or AAV-GCas9. Mutant huntingtin was detected by 1C2 antibody. Uninjected heterozygous HD140Q KI mice were used as a control (scale bar: 20 μm). (F) Quantitative analysis of immunohistochemistry results in (E) (n = 5; ****p < 0.0001; one-way ANOVA with Tukey’s post-tests). Data are represented as mean ± SEM. See also Figure S1.

Figure 3.. Effects of Cas9 Expression on Genes Involved in Neuronal Functions

(A) Schematic representation of stereotaxic viral injection. (B) The mRNA levels of AAV-Cas9 and AAV-GCas9 were compared three weeks after stereotaxic injection via qRT-PCR (n = 3; two-tailed Student’s t test). (C) Heatmap view of 400 significantly changed genes in AAV-Cas9-injected striatal tissues compared with uninjected or AAV-GCas9-injected striatal tissues (n = 3; p < 0.05). (D) Gene ontology (GO) analysis was performed on the 400 significantly changed genes. The top 10 pathways with the lowest false discovery rate (FDR) were shown. (E) qRT-PCR was performed to compare the mRNA levels of various genes selected from the RNA-seq results (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA with Tukey’s post-tests). Data are represented as mean ± SEM. See also Figure S3.

Figure 4.. Validation of RNA-Seq Results

(A) Western blotting analysis was performed to compare the expression of selected proteins in the striatum 3 or 9 weeks after AAV-Cas9 or AAV-GCas9 injection. Vinculin was used as a loading control. (B) Quantitative analysis of western blotting results in (A) (n = 5; *p < 0.05; **p < 0.01; ***p < 0.001; one-way ANOVA with Tukey post-tests). (C) Heatmap view of 94 significantly changed genes in AAV-Cas9-injected striatal tissues compared with AAV-GFP- or AAV-GCas9-injected striatal tissues (n = 4; p < 0.05). (D) GO analysis was performed on the 94 significantly changed genes. The top 10 pathways with the lowest FDR were shown. Data are represented as mean ± SEM. See also Figure S3.