Per 100 µl transfection, 1 µl of 200 µM crRNA and 1 µl 200 µM trans-activating crRNA in duplex buffer (all from Integrated DNA Technologies) were mixed, denatured at 95°C for 5 min, and renatured for 5 min at room temperature. 5.6 µl PBS and 2.4 µl 61 µM Cas9 V3 (1081059; Integrated DNA Technologies) were added and incubated for 15–30 min. If required, RNPs were mixed at the following ratios: 50% crIgH, 25% crIgK1, and 25% crIgK2 (mouse) or 50% crhIgH3 and 50% crhIgK3 (human). 4 µl 100 µM electroporation enhancer in duplex buffer or 4 µl HDRT at 2.5 µg/µl was added to 10 µl mixed RNP and incubated for a further 1–2 min.
24 h after stimulation, activated mouse or human B cells were harvested, washed once in PBS, and resuspended in Mouse B cell Nucleofector Solution with Supplement (murine B cells) or Primary Cell Nucleofector Solution 3 with Supplement (humanB cells) prepared following to the manufacturer’s instructions (Lonza) at a concentration of 4–5 × 106 cells/86 µl. 86 µl cells were added to the RNP/HPRT mix, gently mixed by pipetting, transferred into nucleofection cuvettes, and electroporated using an Amaxa IIb machine setting Z-001 (murine B cells) or Amaxa 4D machine setting EH-140 (human B cells). Cells were immediately transferred into 6-well dishes containing 5 ml prewarmed mouse or human B cell medium supplemented with the relevant anti-RP105 antibody at 2 µg/ml and incubated at 37°C 5% CO2 for 24 h before further processing.