title: Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells.
abstract: Mammalian neural stem cell (NSC) lines provide a tractable model for discovery across stem cell and developmental biology, regenerative medicine and neuroscience. They can be derived from foetal or adult germinal tissues and continuously propagatedin vitroas adherent monolayers. NSCs are clonally expandable, genetically stable, and easily transfectable - experimental attributes compatible with targeted genetic manipulations. However, gene targeting, which is crucial for functional studies of embryonic stem cells, has not been exploited to date in NSC lines. Here, we deploy CRISPR/Cas9 technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NSC lines, including: (1) efficient targeted transgene insertion at safe harbour loci (Rosa26andAAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in of epitope tags and fluorescent reporters (e.g.Sox2-V5andSox2-mCherry); and (4) engineering of glioma mutations (TP53deletion;H3F3Apoint mutations). These resources and optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Raul Bardini
authors.initials: RB
authors.lastname: Bressan
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Pooran Singh
authors.initials: PS
authors.lastname: Dewari
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Kalantzaki
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Ester
authors.initials: E
authors.lastname: Gangoso
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Mantas
authors.initials: M
authors.lastname: Matjusaitis
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Claudia
authors.initials: C
authors.lastname: Garcia-Diaz
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Carla
authors.initials: C
authors.lastname: Blin
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Vivien
authors.initials: V
authors.lastname: Grant
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Harry
authors.initials: H
authors.lastname: Bulstrode
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.
authors.forename: Sabine
authors.initials: S
authors.lastname: Gogolok
authors.affiliation: Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge, UK.
authors.forename: William C
authors.initials: WC
authors.lastname: Skarnes
authors.affiliation: MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK steven.pollard@ed.ac.uk.
authors.forename: Steven M
authors.identifier: 0000-0001-6428-0492
authors.initials: SM
authors.lastname: Pollard
mesh_terms: D000483:Alleles; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D018604:Epitope Mapping; D000939:Epitopes; D018390:Gene Targeting / Q000379:methods*; D005910:Glioma / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D059765:Homologous Recombination; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D058953:Neural Stem Cells / Q000166:cytology*; D000076104:Oligodendrocyte Transcription Factor 2; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation; D011995:Recombination, Genetic; D044968:Regenerative Medicine; D019076:Transgenes
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D000939:Epitopes; D049452:Green Fluorescent Proteins; D009419:Nerve Tissue Proteins; D000076104:Oligodendrocyte Transcription Factor 2; D009841:Oligonucleotides; C512773:Olig2 protein, mouse
keywords: CRISPR/Cas9; Epitope tagging; Gene targeting; Genome editing; Glioblastoma; Homologous recombination; Neural stem cell; Transcription factor