Transfection of TcB transposase mRNA and BAFF-CAR transposon plasmid were achieved using electroporation performed with the 4D-nucleofector (Lonza). Electroporation was performed with the 4D-Nucleofector (Lonza, Basel, Switzerland). T cells were centrifuged to remove the culture medium and resuspended in P3 buffer. For stable BAFF-CAR integration, 2.5 µg of BAFF-CAR transposon backbone. Reactions were set up using the ddPCR Supermix for Probes (no dUTP) (Bio-Rad, Hercules, CA) with 50 ng of genomic DNA per assay and droplets were generated and analyzed with the QX200 Droplet-digital PCR system (Bio-Rad, Hercules, CA).
Pooled CRISPR sgRNA (Synthego) targeting the BAFF receptors (BAFF-R, TACI, BCMA) were introduced into luciferase-expressing Jeko-1 cells or RPMI-8226 cells via nucleofection (Lonza Nucleofector 2b). 1ug of each sgRNA was used in tandem with 1.5 µg of Cas9 mRNA (Sigma-Millipore) into 10e6 cells. Percentage knockout efficiency was measured using flow cytometry 5 days following nucleofection
Primary human cells were sourced from Lonza (Catalog # R-DRG-505),