For each electroporation,150,000-200,000 late log-phase K562 cells were pelleted (100 x g, 5 minutes) and re-suspended in 20 µL Lonza SF solution. 20 µL cells, 10 µL Cas9 RNP containing the desired guide (see above, and Supplementary Table 1), and 1 µL 100 µM ssDNA template programming the desired edit at the SCD SNP were mixed and electroporated using the recommended protocol for K562 cells. After electroporation, K562 cells were incubated for 10 minutes in the cuvette, transferred to 1 mL of K562 media, and cultured for 48-72 h prior to genomic DNA extraction and genotyping.
CD34+: To edit HSCs, ~1 million HSPCs were thawed and cultured in StemSpan SFEM medium supplemented with StemSpan CC110 cocktail (StemCell Technologies) for 24 h prior to electroporation with Cas9 RNP. To electroporate
HSPCs, 100,000-200,000 were pelleted (200 x g, 10 minutes) and resuspended in 20 µL Lonza P3 solution, and mixed with 10 µL Cas9 RNP and 1 µL 100 µM ssDNA template programming the desired edit. This mixture was electroporated using the
Lonza 4d nucleofector and either of two protocols (“1” : DO100, “2”: ER100). Electroporated cells were recovered in the cuvette with 200 µL StemSpan
SFEM/CC110 for 10-15 minutes, and transferred to culture in 1 mL StemSpan SFEM/CC110 for 48 hours post-electroporation.