Pre-activated B cells were transfected using the P4 Primary Cell 4D-Nucleofector X Kit L (Lonza, V4XP-4024) in combination with the program DI-100. The following standard conditions in 100 µl total volume of nucleofectionmix were used, if not described differently: 1 × 106 cells, 20 µg Cas9 protein complexed with 0.156 nmol Alt-R duplex gRNA at 1:1.125 ratio and 5 µg of linearized double-stranded DNA generated by PCR. After electroporation, edited cells were seeded in 5–6mL culturing medium supplemented with rIL-4 into a 6-well plate in the presence of irradiated 40 LB cells (5 × 105 cells per well). Two days after transfection (Day 3), the B cell culturing medium was replaced by carefully aspirating the medium and adding 5ml of fresh B cell medium supplemented with rIL-4. One day later
(Day 4), primary B cells were harvested and prepared for flow cytometry analysis.

Hybridoma cells were electroporated using the SF Cell Line 4DNucleofector X Kit L (Lonza, V4XC-2024) with the program CQ-104. The following standard conditions in 100 µl of total volume of nucleofection mix were used: 1 × 106 cells, 0.156 nmol pre-complexed Alt-R duplex gRNA and 5 µg of PCRlinearized double-stranded DNA. Following transfection, cells were incubated for 5min at RT, before adding 500 µl of prewarmed medium to the nucleocuvette and typically transferring themto 1.5mL of fresh growthmediumin 6-well plates. The cells were usually supplemented 24 h later with 0.5–1.0mL of fresh culturing medium.