Efficient CRISPR/Cas9-assisted gene targeting enables rapid and precise genetic manipulation of mammalian neural stem cells

Abstract

Mammalian neural stem (NS) cell 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 propagated in vitro as adherent monolayers. NS cells are clonally expandable, genetically stable, and easily transfectable – experimental attributes compatible with targeted genetic manipulations. However, gene targeting – so critical for functional studies of embryonic stem cells – has not been exploited to date in NS cells. Here we deploy CRISPR/Cas technology to demonstrate a variety of sophisticated genetic modifications via gene targeting in both mouse and human NS cell lines, including: 1) efficient targeted transgene insertion at safe harbor loci (Rosa26 and AAVS1); 2) biallelic knockout of neurodevelopmental transcription factor genes; 3) simple knockin of epitope tags and fluorescent reporters (e.g. Sox2-V5 and Sox2-mCherry); and 4) engineering of glioma mutations (TP53 deletion; H3F3A point mutations). These resources and optimized methods enable facile and scalable genome editing in mammalian NS cells, providing significant new opportunities for functional genetic analysis.