P3 primary cell 4d nucleofector x kit l

The 4D-Nucleofector™ System (Lonza) was used to electroporate piggyBac and transposase vectors into iNGN cells in suspension (X-Unit, P3 Primary Cell 4D-Nucleofector® X Kit L, program CB-156) following the manufacturer’s guidelines. By default, 10 µg transposon DNA and 2 µg transposase vector (System Biosciences) were applied in less than 10 µl volume to 800k iNGN cells. After nucleofections, cells were kept under constant 20 µg/ml blasticidin (Thermo Fisher Scientific, A1113903) selection.

Cells were treated with 10 μM Rock Inhibitor Y-27632 for 3 h before transfection. IPSC were transfected using a 4D-Nucleofector System (Lonza, Basel, Switzerland) with an electroporation buffer P3 Primary Cell 4D-Nucleofector X Kit L (Lonza, Basel, Switzerland) and program CA137. Cells were detached with Accutase and washed with PBS. Around 8 × 10⁵ cells were mixed with either 7 μg pX358 vector expressing wild-type SpCas9, tracrRNA and gRNA or 3.5 μg of pX461 vector expressing D10A nickase SpCas9, tracrRNA and one gRNA and 3.5 μg of pX461 vector expressing D10A nickase SpCas9, tracrRNA and the other gRNA of the pair to be used with the nickase approach. Donor templates were used as single stranded DNA named DT7.1 for wild-type Cas9-gRNA1 and nickase Cas9-gRNA3a-gRNA3b, or DT7.23 for wild-type Cas9 gRNA2 and nickase Cas9-gRNA4a-gRNA4b. For each reaction, 2 μL of 100 μΜ donor template was used. Sequences of donor template are provided in Supplementary Table S4.
The format of transfection was NucleocuvetteTM Vessels (Lonza, Basel, Switzerland). Each reaction was seeded in one well of 24-well plate vitronectin in mTeSR medium supplemented with 10 μM Rock Inhibitor Y-27632. Cells were enriched based on GFP positivity 24 h post transfection, as described below.

PBMCs were thawed and stimulated with Dynabeads human T-activator CD3-CD28 (11131D; Gibco) at a ratio of three beads for one cell in the presence of IL-2 (30 IU/ml). After 48 h, beads were removed, and the cells were electroporated using a P3 primary cell 4D-Nucleofector X kit L (V4XP-3024; Lonza) and the program EO-115. Before electroporation, RNP complexes were formed by incubating equal volumes of sgRNA (44 µM) and Alt-R S.p. HiFi Cas9 nuclease V3 (36 µM; 1081061; Integrated DNA Technologies) at room temperature for 20 min according to the manufacturer’s instructions. 1.5–3.0 × 10⁶ activated T cells were electroporated with either 10 µl RNP plus 10 µg of HDR DNA template (edited T cells) or Cas9 alone (control T cells). Cells were analyzed by flow cytometry for CD90.1 expression 4 d after transfection using anti–CD4-APC (555349; BD Biosciences), anti–CD8-FITC (555366; BD Biosciences), and anti–CD90.1-PE (202524; BioLegend) antibodies. Edited T cells were sorted into CD4⁺CD90-1⁺ and CD8⁺CD90.1⁺ subpopulations 5 d after transfection using a FACS Aria cell sorter (BD Biosciences). CD4⁺CD90.1⁻ cells and CD8⁺CD90.1⁻ control T cells were sorted in parallel and used as negative controls.

The tightly synchronized mature schizont-stage parasites of P. knowlesi were transfected using the Amaxa 4D electroporator (Lonza, Basel, Switzerland) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) following previous reports (Moon et al., 2016 (link)). Briefly, a 20-μg repair template and 20 μg pCas9/sg (Mohring et al., 2019 (link)) containing sgRNA sequences for pkmsp1p were mixed with P3 Primary Cell nucleofector solution, including supplement 1 (Lonza), and transferred to a Nucleocuvette™ Vessel (Lonza), followed by nucleofection with program FP158. Transfected parasites were immediately transferred to complete media with RBCs and incubated at 550 rpm for approximately 30 min at 37°C to allow invasion before transferring to standard culture conditions. After 24 h, transfected parasites were selected by drug pressure with 100 nM pyrimethamine (Sigma-Aldrich), and the medium, including pyrimethamine, was replaced at daily intervals for 5 days. The transfected parasites were cloned out by limiting dilution and confirmed by genotyping with diagnostic primers of extracted genomic DNA (Supplementary Tables 1, 2).

Cas9 RNPs were prepared immediately before experiments by incubating the crRNA-tracrRNA duplex with the Alt-R S.p. Cas9 Nuclease V3 (IDT) in a 2:1 ratio (2.5 μM Cas9 + 5 μM crRNA-tracrRNA duplex per electroporation) for 20 min at room temperature. For each electroporation, the cells were resuspended in PBS and washed at 200g for 10 min at room temperature. Per electroporation, 10 million cells were resuspended in 87 μl of P3 primary cell nucleofection solution (Lonza) and mixed with 13.5 μl of Cas9 RNP. Cells with the Cas9 RNP were then transferred to the 100 μL Nucleocuvette Vessel (P3 Primary Cell 4D-Nucleofector X Kit L, Lonza) and electroporated using a 4D nucleofector (Lonza) with program EH-115. After nucleofection, cells were left in 100 μL Nucleocuvette Vessel for 10 min at room temperature. Cells were then transferred to a 6-well plate with X-VIVO15 + 5% human AB serum and incubated at 37°C for 4 to 5 h before reactivation.