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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.

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Fig. 1: No significant physiological deficit or cytotoxicity is found in primary cultured neurons after CRISPR–Gold treatment.
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.
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.
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.
Fig. 5: mGluR5–CRISPR successfully promotes mGluR5 gene editing in the striatum of wild-type and Fmr1 knockout mice.
Fig. 6: Knocking out mGluR5 using mGluR5–CRISPR significantly rescues the increased repetitive behaviours in Fmr1 knockout mice.

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Acknowledgements

We thank J. Doudna for advice, B. Staahl for discussions and technical support, and H. Kim, A. Rao and K. Kataoka for technical support. We thank M. A. Bhat and members of the Bhat Lab for technical support. We thank M. West in the CIRM/QB3 Shared Stem Cell facility for technical support. This work was supported by the National Institutes of Health grant R01EB023776 to N.M, and by the National Science Foundation grant 1456862 to R.B.

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B.L., K.L., R.B., N.M. and H.Y.L. designed the research, and B.L., K.L., S.P., R.G.-R., A.C., H.M.P., V.B. and H.Y.L. performed the experiments and analyses. R.G.-R. and S.P. generated the videos. B.L., K.L., N.M. and H.Y.L. wrote the manuscript. H.Y.L. supervised the research. All authors discussed the results and commented on the manuscript.

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Correspondence to Niren Murthy or Hye Young Lee.

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K.L., H.M.P. and N.M. are co-founders of GenEdit Inc. The remaining authors declare no competing interests.

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Supplementary Video 1

Marble-bury assay.

Supplementary Video 2

Empty-cage observations.

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Lee, B., Lee, K., Panda, S. et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat Biomed Eng 2, 497–507 (2018). https://doi.org/10.1038/s41551-018-0252-8

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