Optimized CRISPR Editing in B Cell Lines

For cell lines, ImProB^{15 (link)}, A-20^{31 (link)}, Ramos, and i.29 IgA^{32 (link)} electroporations were performed with 18.3pmol Cas9 and 22pmol gRNA, at 1.0E5cells/μl in OptiMEM (31985-047, gibco) using 10 μl tips in a Neon electroporation system (Invitrogen). For the human cell line, parameters were: 1350 v 30 ms 1 pulse and for the mouse cell lines: 1600v 20 ms, 1 pulse. All cell lines were grown in 1640 RPMI (01-100-1A, Biological Industries) supplemented with 10% HI FBS (04-127-1A, Biological Industries), 50 μM β-Mercaptoethanol and P/S (03-031-1B, Biological Industries). Transductions were performed with a 50,000 MOI of rAAV-DJ for mouse cell lines, and a 130,000 MOI of rAAV-DJ for human cell line. Efficiency of editing was determined 3 days following electroporation.
For plasmid electroporations we used 3 µg plasmid DNA/1E06 cells/10 µl Neon tip.
The Ramos cell line (ATCC CRL-1596) was provided by the Benhar lab, Tel Aviv University. The i.29 cell line was provided by the Zan Bar lab, Tel Aviv University. The ImProB cell line was provided by the Deriano lab, Pasteur Institute. The A-20 cell line was provided commercially (ATCC TIB-208).