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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.

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