All cell transfections were performed with a Nucleofector (Lonza) according to the manufacturer’s 96-well shuttle protocol for respective cell lines. After trypsinization, cells were counted, pelleted, and washed twice in Hanks balanced salt solution to minimize nuclease carryover from growth medium. Specifically, for the rat C6 cell line, SG solution and program FF-127 were used to transfect 200,000 cells. For the mouse Neuro-2a cell line, SF solution and program 96-DS-137 were used to transfect 200,000 cells per reaction. One microgram of a GFP plasmid was always transfected for each conditionas a control. ZFN mRNAs were tranfected at 2 µg/reaction. Regardless of the delivery method of Cas9 (plasmid, mRNA or protein), sgRNAs were used at 2–12 µg/reaction. Cas9 expression plasmid was transfected at 2 µg/reaction, Cas9 mRNA at 2–4 µg/reaction, and recombinant Cas9 protein at 3–10 µg/reaction.