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.