16 well nucleocuvette strip

K562 and primary T cells were electroporated using a Lonza 4D-Nucleofector device (Lonza). The SF Cell Line 4D X Kit S (Lonza) was used for K562s and the P3 Primary Cell 4D Kit (Lonza) was used for the unstimulated primary T cells. Approximately 1.5e6 K562 cells were electroporated in a final volume of 20 μL in a 16-well nucleocuvette strip (Lonza). For the T-cell experiments, 7.25e6 primary T cells were electroporated in a final volume of 100 μL in a cuvette.
For the single vector and two vector PASTE systems delivered to K562 cells, 900 ng of prime-Bxb1 complex-encoding plasmid or 800 ng of prime-encoding plasmid and 100 ng of Bxb1-encoding plasmid were electroporated, respectively. For both systems, 250 ng cargo plasmid, 200 ng atgRNA guide-encoding plasmid and 80 ng RNA nicking guide-encoding plasmid were added.
For T-cell electroporations, 990 ng of a guide vector expressing both the atgRNA and nicking guide, 875 ng of the EGFP-containing minicircle plasmid, and 3,150 ng of the PASTE plasmid (SpCas9-RT-P2A-Bxb1) were electroporated.
Electroporations were performed according to the manufacturer’s protocol and after 72 hours the cells were harvested for genomic DNA isolation and digital droplet PCR quantification.

Nucleofection was performed on K562, HeLa, and U2OS cells as previously described^{38 (link)}. 750ng of base editor-expression plasmid and 250ng sgRNA-expression plasmid were nucleofected in a final volume of 20uL in a 16-well nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 10⁵ cells per sample (program FF-120), according to the manufacturer’s protocol. U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 3–4 × 10⁵ cells per sample (program DN-100), according to the manufacturer’s protocol. HeLa cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 10⁵ cells per sample (program CN-114), according to the manufacturer’s protocol. Nucleofiection of HAP1 cells was performed using the same amounts of DNA and final volume in a 16-well nucelocuvette strip; however, HAP1 cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 4 × 10⁵ cells per sample (program DZ-113), according to the manufacturer’s protocol. Cells were harvested 72 hours after nucleofection for genomic DNA extraction.

We combined 500 ng of Left DdCBE monomer and 500 ng of Right DdCBE monomer in a volume that did not exceed 2 μl. This combined plasmid mixture was nucleofected in a final volume of 22 μl per sample in a 16-well Nucleocuvette strip (Lonza). U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 30,000–50,000 cells per sample (program DN-100), according to the manufacturer’s protocol.