Nucleocuvette plate

Electroporation was performed using the Lonza™ Nucleofector™ 96-well Shuttle™ System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1× phosphate buffered saline (PBS) and resuspended in 20 µL of solution SF or SE (Lonza). Cell suspensions were combined with RNP complex(es), Alt-R Cas9 or Cpf1 (Cas12a) Electroporation Enhancer (Integrated DNA Technologies) and HDR donor template (if applicable). This mixture was transferred into one well of a Nucleocuvette™ Plate (Lonza) and electroporated using manufacturer’s recommended protocols (except for HEK293, which used protocol 96-DS-150). After nucleofection, 75 µL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 µL was transferred to a 96-well culture plate with 175 µL pre-warmed culture media. Transfection plates were incubated at 37 °C and 5% CO₂.

Electroporation was performed using the Lonza Nucleofector 96-well Shuttle System (Lonza, Basel, Switzerland). For each nucleofection, cells were washed with 1× PBS and resuspended in 20 μL of solution SF or SE (Lonza). HAP1 experiments used ~350,000 cells per nucleofection, and Jurkat experiments used ~500,000 cells per nucleofection. Then, cell suspensions were combined with an RNP complex. For Cas9, the RNP concentration was 4 μM with 4 μM Alt-R Cas9 Electroporation Enhancer. For Cas12a, the RNP concentration was a suboptimal dose of 0.2 μM with 3 μM Alt-R Cas12 Electroporation Enhancer (Integrated DNA Technologies) to provide a more diverse range of editing frequencies. This mixture was transferred into one well of a Nucleocuvette Plate (Lonza) and electroporated using manufacturer’s recommended protocols. After nucleofection, 75 μL pre-warmed culture media was added to the cell mixture in the cuvette, mixed by pipetting, and 25 μL was transferred to a 96-well culture plate with 175 μL pre-warmed culture media. Transfection plates were incubated at 37°C and 5% CO₂.

K562 cells (ATCC, CCL243) were cultured using RPMI-1640 growth medium supplemented with 10% FBS (Fetal Bovine Serum) and 1x PSG (Penicillin-Streptomycin-Gentamycin, Gibco, 10378-016) and maintained at 37 °C with 5% CO₂. ZF-CBEs and GFP were dosed as plasmid DNA (pDNA) in K562 cells. K562 cells were electroporated with pDNA using the SF cell line 96-well Nucleofector kit (Lonza, V4SC-2960) using manufacturer’s protocol. Prior to electroporation, K562 cells were centrifuged at ~300 × g for 5 min. Cells were resuspended at 2e5 cells per 12 µl of supplemented SF cell line 96-well Nucleofector solution. Twelve µl of cells were mixed with 8 µl of pDNA (400 ng of each ZF-CBE-L, ZF-CBE-R and optionally the nickase construct) and transferred to the Lonza Nucleocuvette plate. Nucleofector program 96-FF-120 was used to electroporate K562 cells with the pDNA mix on the Amaxa Nucleofector 96-well Shuttle System (Lonza). After electroporation, cells were incubated for 10 min at room temperature and transferred to a 96-well tissue culture plate containing 180 µl of complete medium (prewarmed to 37 °C). K562 cells were incubated for ~72 h and then harvested for base editing and indel quantification.