title: CRISPR/Cas9-induced disruption of gene expression in mouse embryonic brain and single neural stem cells in vivo.
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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Nereo
authors.initials: N
authors.lastname: Kalebic
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Taverna
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Stefania
authors.initials: S
authors.lastname: Tavano
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Fong Kuan
authors.initials: FK
authors.lastname: Wong
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Dana
authors.initials: D
authors.lastname: Suchold
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany.
authors.forename: Sylke
authors.initials: S
authors.lastname: Winkler
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany huttner@mpi-cbg.de sarov@mpi-cbg.de.
authors.forename: Wieland B
authors.initials: WB
authors.lastname: Huttner
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Dresden, Germany huttner@mpi-cbg.de sarov@mpi-cbg.de.
authors.forename: Mihail
authors.initials: M
authors.lastname: Sarov
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D058953:Neural Stem Cells / Q000378:metabolism*; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods; D013687:Telencephalon / Q000166:cytology* / Q000196:embryology; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C489435:Btg2 protein, mouse; D017874:Immediate-Early Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D025521:Tumor Suppressor Proteins
keywords: CRISPR/Cas9; in utero electroporation; microinjection; neural stem cell; neurogenesis