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

Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, Gogolok S, Skarnes WC, Pollard SM.
Source: Development
Publication Date: (2017)
Issue: 144(4): 635-648
Research Area:
Cancer Research/Cell Biology
Stem Cells
Gene Expression
Cells used in publication:
Neural stem cell (NSC), mouse
Species: mouse
Tissue Origin: brain
Neural stem cell (NSC), human
Species: human
Tissue Origin: brain
Nucleofector™ I/II/2b
4D-Nucleofector™ X-Unit
Cells were transfected using either Amaxa 2B or 4D nucleofection systems (Lonza) according to the manufacturer’s instructions. For the 2B system, 2×106 cells were pre-mixed with plasmid DNA, 2 µg of the indicated Cas9 vector, 1 µg of each sgRNA and 1 µg of the targeting vector or 2 µg of ssODN in 100 µl of Neural Stem Cell Amaxa nucleofection buffer. Nucleofection program T-030 and X-005 was used for mouse and human NSCs, respectively. For the 4D system, 16-strip cuvettes were loaded with, unless otherwise stated, 4×105 cells and 0.8 µg plasmid DNA (0.4 µg Cas9, 0.1 µg each sgRNA and 0.2 µg targeting vector) in SG transfection solution (Lonza). Program DN100 gave best survival and transfection efficiency for plasmid DNA delivery. For delivery of the Cas9/sgRNA complex, 5 µg (unless otherwise stated) of recombinant Cas9 were mixed with 3 µg of in vitro transcribed sgRNA and allowed to form ribonucleoprotein complex at room temperature for 10 min. The Cas9/sgRNA complex together with 1.5 µg of ssODNwas transfected into 2×105 cells using theAmaxa 4D 16-strip cuvettes in SG transfection buffer. Program EN138 gave the best results for rCas9/IVT sgRNA delivery. After transfection, cells were recovered in pre-warmed culture media and plated onto 10 cm culture dishes for 5 days prior to drug selection or downstream assays.
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.