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).