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EMBO Rep. 2016 Mar;17(3):338-48. doi: 10.15252/embr.201541715. Epub 2016 Jan 12.

CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.

Author information

1
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
2
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany huttner@mpi-cbg.de sarov@mpi-cbg.de.

Abstract

We have applied the CRISPR/Cas9 system in vivo to disrupt gene expression in neural stem cells in the developing mammalian brain. Two days after in utero electroporation of a single plasmid encoding Cas9 and an appropriate guide RNA (gRNA) into the embryonic neocortex of Tis21::GFP knock-in mice, expression of GFP, which occurs specifically in neural stem cells committed to neurogenesis, was found to be nearly completely (≈ 90%) abolished in the progeny of the targeted cells. Importantly, upon in utero electroporation directly of recombinant Cas9/gRNA complex, near-maximal efficiency of disruption of GFP expression was achieved already after 24 h. Furthermore, by using microinjection of the Cas9 protein/gRNA complex into neural stem cells in organotypic slice culture, we obtained disruption of GFP expression within a single cell cycle. Finally, we used either Cas9 plasmid in utero electroporation or Cas9 protein complex microinjection to disrupt the expression of Eomes/Tbr2, a gene fundamental for neocortical neurogenesis. This resulted in a reduction in basal progenitors and an increase in neuronal differentiation. Thus, the present in vivo application of the CRISPR/Cas9 system in neural stem cells provides a rapid, efficient and enduring disruption of expression of specific genes to dissect their role in mammalian brain development.

KEYWORDS:

CRISPR/Cas9; in utero electroporation; microinjection; neural stem cell; neurogenesis

PMID:
26758805
PMCID:
PMC4772980
DOI:
10.15252/embr.201541715
[Indexed for MEDLINE]
Free PMC Article
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