Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours

Abstract

Technologies that can safely edit genes in the brains of adult animals may revolutionize the treatment of neurological diseases and the understanding of brain function. Here, we demonstrate that intracranial injection of CRISPR-Gold, a nonviral delivery vehicle for the CRISPR-Cas9 ribonucleoprotein, can edit genes in the brains of adult mice in multiple mouse models. CRISPR-Gold can deliver both Cas9 and Cpf1 ribonucleoproteins, and can edit all of the major cell types in the brain, including neurons, astrocytes and microglia, with undetectable levels of toxicity at the doses used. We also show that CRISPR-Gold designed to target the metabotropic glutamate receptor 5 (mGluR5) gene can efficiently reduce local mGluR5 levels in the striatum after an intracranial injection. The effect can also rescue mice from the exaggerated repetitive behaviours caused by fragile X syndrome, a common single-gene form of autism spectrum disorders. CRISPR-Gold may significantly accelerate the development of brain-targeted therapeutics and enable the rapid development of focal brain-knockout animal models.

Fig. 1 |. No significant physiological deficit or cytotoxicity is found in primary cultured neurons after CRISPR–Gold treatment.

a, Schematic of CRISPR–Gold synthesis. DNA oligonucleotide-conjugated GNPs bind to Cas9 or Cpf1 RNPs, and subsequent PAsp(DET) polymer encapsulation generates CRISPR–Gold. b–d, Primary cultured neurons (days in vitro 7 (DIV7)) were treated with CRISPR–Gold Cas9 RNPs (CRISPR–Gold) and were compared with untreated neurons (control) for electrophysiological properties by whole-cell current clamp recording. Neurons were measured for membrane potential (b), input resistance (c) and the number of spikes generated by a 200pA current injection (d). n = 20–21 for control, n = 15–17 for CRISPR–Gold, mean ± s.e.m. No significant differences in the membrane potentials, input resistance or the number of spikes were found between the groups. e, Representative traces for control and CRISPR–Gold. f, Left: DIV7 primary cultured neurons were treated with CRISPR–Gold Cas9 RNPs (CRISPR–Gold) and were compared with untreated neurons (control). Neurons were fixed 14 days after CRISPR–Gold treatment and stained with SYTOX Red to identify dead cells (red) and with phalloidin-Alexa 488 for visualizing neuronal morphology (green). Scale bar, 100 μm. Right: quantification of SYTOX⁺ cells (%) among DAPI⁺ cells in the control and CRISPR–Gold groups. n = 6 for control, n = 5 for CRISPR–Gold, mean ± s.e.m. No significant difference in the percentage of SYTOX⁺ cells was found between groups. This experiment was replicated twice.

Fig. 2 |. YFP expression is efficiently reduced in the neurons of the mouse brain using CRISPR–Gold delivery of Cas9 or Cpf1 RNPs in Thy1-YFP mice.

a, Schematic of CRISPR–Gold delivery of Cas9 or Cpf1 RNPs into the brains of Thy1-YFP mice. b, Left: schematic of Cas9 or Cpf1 RNP-mediated indel mutation. Red arrow indicates the target region for gene-editing. Right: schematic of stereotaxic injection into the hippocampus (Bregma: −2.18 mm) of Thy1-YFP mice using the CRISPR–Gold system. c,d, Left: immunostaining of YFP-labelled neurons (green) with nuclei staining with DAPI (blue) 2 weeks after stereotaxic injection of Cas9 (c) or Cpf1 (d) RNPs using the CRISPR–Gold system into the dentate gyrus of the hippocampus of Thy1-YFP mice. Scale bars, 100 μm. Right: quantification of the YFP⁺ cells normalized to DAPI⁺ cell numbers in the granule cell layer of the dentate gyrus in the injected side (CRISPR–Gold) compared with the contralateral control side (control). n = 6 for each group, mean ± s.e.m., ****P < 0.0001 compared with the control side, Student’s unpaired t-test. This experiment was replicated four times.

Fig. 3 |. Deletion of stop sequences and expression of tdTomato in the brain of Ai9 mice by CRISPR–Gold delivery of Cas9 or Cpf1 RNPs into the hippocampus.

a, Schematic of CRISPR–Gold delivery of Cas9 or Cpf1 RNPs into the brains of Ai9 mice. b, Left: schematic of Cas9 or Cpf1 RNP-mediated deletion. Yellow arrowheads represent the 5′ and 3′ ends of the stop sequence. Red arrows indicate target regions for gene editing. Right: schematic of stereotaxic injection into the hippocampus (Bregma: −2.18 mm) of Ai9 mice using the CRISPR–Gold system. c,d, Left: immunostaining of tdTomato (red) and nuclei staining with DAPI (blue) 2 weeks after stereotaxic injection of Cas9 (c) or Cpf1 (d) RNPs using the CRISPR–Gold system into the hippocampus of Ai9 mice. The uninjected side (control) and the injected side (CRISPR–Gold) are shown in the upper panels. Scale bars, 200 μm. Higher-magnification images of the injected side (yellow box) are shown in the lower panels. Scale bars, 100 μm. Right: quantification of the percentage of tdTomato⁺ cells among DAPI⁺ cells in the Cas9 RNP-injected area (c) and the Cpf1 RNP-injected area (d). n = 11–12 for each group, mean ± s.e.m., ****P < 0.0001 compared with the control side, Student’s unpaired t-test. This experiment was replicated twice.

Fig. 4 |. Deletion of stop sequences and expression of tdTomato in the brain of Ai9 mice by CRISPR–Gold delivery of Cas9 or Cpf1 RNPs into the striatum.

a, Left: schematic of Cas9 or Cpf1 RNP-mediated deletion. Yellow arrowheads represent the 5′ and 3′ ends of the stop sequence. Red arrows indicate target regions for gene editing. Right: schematic of stereotaxic injection into the striatum (Bregma: 0.26 mm) of Ai9 mice using the CRISPR–Gold system. b,c, Left: immunostaining of tdTomato (red) and nuclei staining with DAPI (blue) 2 weeks after stereotaxic injection of Cas9 (b) or Cpf1 (c) RNPs using the CRISPR–Gold system into the striatum of Ai9 mice. The uninjected side (control) and the injected side (CRISPR–Gold) are shown in the upper panels. Scale bars, 400 μm. Higher-magnification images of the injected side (yellow box) are shown in the lower panels. Scale bars, 200 μm. Right: quantification of the percentage of tdTomato⁺ cells among DAPI⁺ cells in the Cas9 RNP-injected area (b; n = 14 for each group, mean ± s.e.m.) and Cpf1 RNP-injected area (c; n = 12–13 for each group, mean ± s.e.m.). ****P < 0.0001 compared with the control side, Student’s unpaired t-test. This experiment was replicated twice. d,e, Quantification of the number of DAPI⁺ cells in the Cas9 (d) or Cpf1 RNP-injected area (striatum) (e). Two weeks after stereotaxic injection of Cas9 or Cpf1 RNPs using the CRISPR–Gold system into the striatum of Ai9 mice, the brains were sliced and immunostained with tdTomato antibodies and stained with DAPI. The number of DAPI⁺ cells of equal size in the injected ROI of the striatum in both the control group and the CRISPR–Gold group were compared and analysed. n = 6 for each group, mean ± s.e.m., NS, not significant, Student’s unpaired t-test. This experiment was replicated twice.

Fig. 5 |. mGluR5–CRISPR successfully promotes mGluR5 gene editing in the striatum of wild-type and Fmr1 knockout mice.

a, Upper: schematic of the injection process for mGluR5–CRISPR into the striatum of wild-type (WT) and Fmr1 knockout (KO) mice. Saline or mGluR5–CRISPR was injected into the striatum (Bregma: 0.26 mm, 3 injection sites per hemisphere are indicated as blue dots, 0.4-mm interval) of WT or Fmr1 KO mice. Lower: schematic of the target sequences of Cas9 RNPs and the protospacer adjacent motif (PAM) for Grm5 knockout. b, RNA was extracted from the saline-injected control side (control) or from the mGluR5–CRISPR-injected side (mGluR5–CRISPR) of WT or Fmr1 KO mice 11 weeks after stereotaxic injections. mRNA levels of Grm5 were amplified and analysed by RT-qPCR. Fold-change of Grm5 mRNA levels are shown after normalization against PPIA mRNA levels. n = 4–6, mean ± s.e.m., ***P < 0.001, ****p < 0.0001, one-way ANOVA. c, Left: immunostaining of mGluR5 (cyan) 5 weeks after stereotaxic injection of saline (control) or mGluR5–CRISPR into the striatum of WT or Fmr1 KO mice. Scale bar, 100 μm. Right: the number of mGluR5⁺ cells in WT control, WT mGluR5–CRISPR, Fmr1 KO control and Fmr1 KO mGluR5–CRISPR groups were counted and normalized to the number of DAPI⁺ cells. n = 8–10, mean ± s.e.m., ****P < 0.0001 by one-way ANOVA. P values were calculated between WT control and Fmr1 KO control, WT control and WT mGluR5–CRISPR, or Fmr1 KO control and Fmr1 KO mGluR5–CRISPR.

Fig. 6 |. Knocking out mGluR5 using mGluR5–CRISPR significantly rescues the increased repetitive behaviours in Fmr1 knockout mice.

a–c, Three weeks after stereotaxic injection of either saline (control) or mGluR5–CRISPR into the striatum of WT and Fmr1 KO mice, the marble-burying assay (a) or the empty cage observation test (b,c) was performed. a, Left: percentage of marbles buried after 30 min of the marble-burying test. Right: representative images after marble-burying assay for 30 min. Jumping (b) and line crossing behaviours (c) were scored during 10 min of an empty cage observation test. n = 10–12 for each group, mean ± s.e.m., *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA. P values were calculated between WT control and Fmr1 KO control, WT control and WT mGluR5–CRISPR, or Fmr1 KO control and Fmr1 KO mGluR5–CRISPR.