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 propagated in vitro as 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 (Rosa26 and AAVS1); (2) biallelic knockout of neurodevelopmental transcription factor genes; (3) simple knock-in 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 optimised methods enable facile and scalable genome editing in mammalian NSCs, providing significant new opportunities for functional genetic analysis.

Elevated FOXG1 and SOX2 in glioblastoma enforces neural stem cell identity through transcriptional control of cell cycle and epigenetic regulators

“…To directly test the functional significance of binding at the Foxo3 CIE, we took advantage of CRISPR/Cas9 genome editing, which we optimized for mouse and human NS cells (Bressan et al 2017). Using a pair of guide RNAs (gRNAs), we deleted the 780-base-pair (bp) Foxg1/ Sox2-bound CIE in FOXG1/SOX2-overexpressing FS3 cells (Fig.…”

“…To test the consequences of Foxo3 deletion, we excised exon 2 of Foxo3 in FS3 cells using CRISPR/Cas9-assisted gene targeting (Bressan et al 2017). Biallelic mutant lines were generated through simultaneous replacement of one Foxo3 allele with an EF1a-puromycin resistance cassette and insertion-deletion (indel) mutations on the remaining allele (Supplemental Fig.…”

“…CRISPR/Cas9 provides new opportunities for decisive functional genetic studies in primary human GBM stem cells. Using recently optimized protocols (Bressan et al 2017), we next performed gene targeting via homologous recombination to delete FOXG1 in human primary GNS cells (G7) (Supplemental Fig. S7A).…”

“…Previous studies have suggested that Sox2 is required for self-renewal of forebrain NS cells: Homozygous knockout by conditional deletion or CRISPR/Cas9 targeting is incompatible with colony formation (Gómez-López et al 2011;Bressan et al 2017). Here, CRISPR/Cas9 was used to mutate the single coding exon of SOX2 (Supplemental Fig.…”

Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis

“…Using CRISPR/Cas9 genome editing approach12, we generated human iPSC mutations in candidate genes identified from the functional genomic studies, to validate and identify novel mechanisms involved in limiting Chlamydia growth in macrophages.…”