Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal peptides and an HA tag at the C-terminus was expressed in Rosetta2 DE3 (UC Berkeley Marcolab) cells, Cas9-NLS was obtained from the QB3 MacroLab at UC Berkeley. 

sgRNAs were synthesized by Synthego as modified gRNAs with 2'-O-methyl analogs and 3' phosphorothioate internucleotide linkages at the first three 5' and 3' terminal RNA residues using protospacer sequences.

crRNAs/tracrRNAs were chemically synthesized (Edit-R, Dharmacon Horizon) using protospacer sequences. ssDonors were obtained by ordering unmodified Ultramer oligonucleotides (IDT). dsDonor was obtained by purifying plasmid DNA from bacterial cultures containing the indicated plasmid (Qiagen) or by SPRI purification of long double-stranded PCR amplicons.

Cas9 RNP assembly and nucleofection: Fifty pmoles of sgRNA was diluted using Cas9 buffer (20?nM HEPES [pH 7.5]), 150mM KCl, 1mM MgCl2, 10% glycerol, and 1mM TCEP) or water. 1.25µL of 40mM Cas9-2xNLS (50 pmoles) was slowly mixed in, and the resulting mixture was incubated for 5min at room temperature to allow for RNP formation. After incubation, either 0.5µL of 100µM ssDonor or 1.5 or 2µg of plasmid DNA was introduced and mixed by pipetting. The total volume of RNP solution was 5ml, where the volume of Cas9 buffer was adjusted to account for volume differences between ssDonor and plasmid DNA. Between 1e+05 and 2e+05 cells were harvested, washed once in PBS, and resuspended in 15µL of nucleofection buffer (Lonza, Basel, Switzerland). Five microliter of RNP mixture was added to 15µL of cell suspensions. Reaction mixtures were electroporated in Lonza 4D nucleocuvettes, incubated in the nucleocuvette at room temperature for five minutes, and transferred to culture dishes containing pre-warmed media. Large-scale nucleofections were performed by splitting cultures and conducting multiple parallel nucleofections.

Editing outcomes were measured four days post-nucleofection by flow cytometry or by amplicon sequencing. Resuspension buffer and electroporation conditions were the following from each cell line: K562 in buffer SF with FF-120, HEK293 in buffer SF with DS-150, T cells in buffer P3 with EH-115, primary mouse glial cells in buffer P3 with DS-112, HDLECs in buffer P3 with CA-137 HCT116s in buffer SE with EN-113, and HeLa cells in buffer SE with CN-114,U251 cells in buffer SE with DS130, iPSCs in buffer P3 with DS-138, HDFs in buffer P3 with DS-150 and HSPCs in buffer P3 with ER-100.