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. 2017 Feb 16;100(3):454–472. doi: 10.1016/j.ajhg.2017.01.030

Figure 4.

Figure 4

In Utero Somatic Genome Editing of Tsc1 and Tsc2 Recapitulates the Pathology and Symptoms Observed in Individuals with FCDII

(A) Schematic figure of the procedure used to generate the mouse model of brain somatic mutations induced by in utero somatic genome editing in the developing brain. In utero electroporation of CRISPR-Cas9 vectors with a dsRed reporter targeting Tsc1 or Tsc2 was performed at E14, and properly electroporated and delivered mice were screened at birth (P0). Then, they were monitored by video-electroencephalography (EEG) after 3 weeks of age. The arrow indicates the focal expression of the dsRed reporter in the embryonic mouse brain. Scale bar, 3 mm.

(B) In utero electroporation of CRISPR vectors expressing selected sgRNA disrupts neuronal migration in the developing mouse neocortex. The images show coronal sections of mouse brains (>P56) electroporated with the CRISPR construct. We counted the percentage of dsRed (+) cells by dividing the number of dsRed (+) cells by the total number of cells (n = 2∼3 per group). Scale bars, 250 μm.

(C) Bar charts showing the relative fluorescence intensities reflecting the distribution of electroporated cells within the cortex. Mice electroporated with the CRISPR construct without sgRNA expression served as controls. Data represent the mean ± SEM (n = 3–6 per group). ∗p < 0.05 and ∗∗∗p < 0.001 compared with the control (two-way ANOVA with a Bonferroni multiple-comparison test).

(D) The EEG wave pattern in the ictal phase of Tsc1 and Tsc2 focal knockout (fKO) mice. EEG signals were recorded from four epidural electrodes located on the left frontal lobe (LF), right frontal lobe (RF), left temporal lobe (LT), and right temporal lobe (RT). Magnified EEG waves of the ictal phase and postictal period are presented in Figure S12.

(E) The seizure frequency in genome-edited mice was measured. Control mice were transfected with a CRISPR construct without sgRNA. Data represent the mean ± SEM (n = 3–6 per group).

(F) The seizure frequency in mice carrying the CRISPR construct targeting Tsc2 was dramatically reduced by rapamycin treatment. Data represent the mean ± SEM (n = 4–12 per group). ∗p < 0.05 and ∗∗p < 0.01 compared with the control (one-way ANOVA with a Bonferroni post-test).