CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells.

Dever DP, Scharenberg SG, Camarena J, Kildebeck EJ, Clark JT, Martin RM, Bak RO, Tang Y, Dohse M, Birgmeier JA, Jagadeesh KA, Bejerano G, Tsukamoto A, Gomez-Ospina N, Uchida N, Porteus MH.
Source: iScience
Publication Date: (2019)
Issue: 15: 524-535
Research Area:
Stem Cells
Cells used in publication:
Neural stem cell (NSC), human
Species: human
Tissue Origin: brain
4D-Nucleofector™ X-Unit

P3 solution and program CA-137: in detail : For experiments with chemically modified sgRNAs and Cas9 mRNA, 500,000 NSCs were electroporated with 15µg Cas9 mRNA and 10µg MS or MSP sgRNA. After nucleofection, NSCs were plated in culturing flasks and cultured for multiple passages.Genome Editing of NSCs with Cas9 mRNA and AAV6 donors A single cell suspension of 500,000 NSCs were responded with 15µg of Cas9 mRNA was mixed with 10µg of sgRNA in 20 uL of P3 solution (Lonza). Nucleofection was performed using 16-well Nucleocuvette Strip with the 4D Nucleofector system (Lonza)  using CA137 code. Immediately after electroporation, cells were transferred into one well of a 48 well plate containing 250 µl of NSC media. Then the HBB UbC-GFP AAV6 donor vector (purchased from Vigene Biosciences) was added directly to the electroporated cells at vector genomes/cell of 10,000, 100,000, or 500,000. After 24 hours, cells were transferred into a T25 flask with 5ml of NSC media and cells were harvested and gDNA was extracted 6 days later (7 days total post electroporation).


Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders