Lentiviral transduction of single guide CRISPR–Cas9 vectors

Pre‐validated CRISPR guides to blood group genes in the lentiviral vector lentiCRISPRv2 were ordered from GenScript (Sanjana et al, 2014). gRNA sequences used are listed in Table EV2. Lentivirus was prepared according to previously published protocols (Satchwell et al, 2015). For transduction of BEL‐A cells, virus was added to 2 × 10⁵ cells in 2 ml medium in the presence of 8 μg/ml polybrene for 24 h. Cells were washed, and resuspended in fresh medium, and after 24 h, transduced cells were selected using 1 μg/ml puromycin for 48 h. Following further expansion, cells were sorted based on antigen null phenotype to derive clones.