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CRISPR /Cas9‐induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo
Author(s) -
Kalebic Nereo,
Taverna Elena,
Tavano Stefania,
Wong Fong Kuan,
Suchold Dana,
Winkler Sylke,
Huttner Wieland B,
Sarov Mihail
Publication year - 2016
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.15252/embr.201541715
Subject(s) - electroporation , neurogenesis , biology , crispr , neural stem cell , microbiology and biotechnology , zebrafish , embryonic stem cell , stem cell , microinjection , progenitor cell , neocortex , gene , genetics , neuroscience
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.