Cas9 RNP assembly and nucleofection:
Cas9 RNP was prepared immediately before experiment by incubating with sgRNA at 1:1.2 molar
ratio in 20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM MgCl2, 10% glycerol and 1 mM TCEP at 37°C for
10 min. HDR template was then added to the RNP mixture. Cells were dissociated by 0.05% trypsin,
spun down by centrifugation at 400×g for 3 min, and washed once with DPBS. Nucleofection of
HEK293T cells was performed using Lonza (Allendale, NJ) SF cell- kits and program CM130 in an
Amaxa 96-well Shuttle system. The human neoFB were transfected with Lonza P2 kit and program
CA137. The hES cells were transfected with P3 primary cell kit and program CB150. Each nucleofection
reaction consisted of approximately 2 × 105 cells in 20 µl of nucleofection reagent and mixed with
10 µl of RNP:DNA. After electroporation, 100 µl of growth media was added to the well to transfer
the cells to tissue culture plates. The cells were incubated at 37°C for 24 hr...