P3 primary cell solution

Direct electroporation of P. knowlesi schizont-infected RBCs was performed as described by Janse et al. [15 (link)] and Moon et al. [9 (link)]. For each electroporation experiment, a total of 1 × 10⁷ or 3.5 × 10⁷ schizont-infected RBCs was purified by Percoll gradient from a 20 mL synchronized P. knowlesi culture (5% hct). The recovered cells (up to 50 μL) were suspended in 100 μL of P3 primary cell solution (Lonza, Basel, Switzerland) and mixed with 20 μg of plasmid DNA in 10 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). This suspension was transferred to a 4D Nucleofector XL cuvette and subjected to electroporation in the Lonza 4D Nucleofector System (Lonza), using program FP158. Immediately after electroporation, the suspension was placed into a 2 mL tube containing 500 μL of cRPMI and 300 μL of stock 50% hct rhesus RBCs. After 30 min at 37°C in a shaking incubator (250 rpm), the mixture (900 μL total) was transferred to a 25 cm² vented culture flask (Thermo Fisher, Waltham, MA, USA) with 5 mL of fresh cRPMI under a 90% N₂/5% CO₂/5% O₂ gas mixture. For drug selection experiments, an additional 200 μL of fresh 50% hct rhesus RBCs was added to cultures. Cells were collected 48 h later for luciferase assays or maintained in culture for selection under 1 nM WR99210 pressure (Fig. 1B).

70–80% confluent iPSC were dissociated into single cells using Accutase (Thermo Scientific, cat # A1110501) and counted using a TC20 Automated Cell Counter (Bio-Rad). iPS cells (8x10⁵ per sample) were pelleted at 1000 rpm for 3 min and cell pellet were gently resuspended in 100 μl of P3 Primary Cell Solution (Lonza, PBP3–02250). Immediately prior to nucleofection, 2 μl (100 pmol/μl) of ssDO was added to 5 μl of pre-assembled Cas9/RNP. One hundred μl of iPS cells resuspended in P3 Primary Cell Solution were transferred to the tube containing Cas9/RNP complexes and ssDO. Cells were mixed twice with Cas9/RNP/Donor oligo and the mixture was transferred to the 100 μl nucleocuvette (Lonza; 2022–01). iPSC were nucleofected immediately using the ‘Primary Cell P3′ program and ‘CA-137′ pulse code. After nucleofection, iPSC were transferred using a Lonza disposable Pasteur pipette into one well of a Matrigel-coated 6-well plate containing 3 mL E8 media with 10 μM Rock inhibitors. Cells were cultured in a 32°C/5% CO₂ incubator for two days and then transferred to a 37°C incubator. Edited iPSC pools were expanded, frozen, and used for generation of clones from single cells.