CRISPR/Cas9-Mediated In Vitro Mutagenesis in GC-Like B Cells.

The CRISPR/Cas9 technology has developed into a powerful tool for genome editing, both in terms of gene silencing and the insertion of precise mutations. However, the application of CRISPR/Cas9-mediated mutagenesis in primary immune cells, in particular in B cells, is still in its infancy because of the difficulty to deliver the CRISPR/Cas9 system into these cells. Here, we describe a new method to use CRISPR/Cas9 for manipulating genes in germinal center (GC)-like B cells in vitro. We isolated Cas9-expressing B cells from R26-Cas9iGFP/+ mice (expressing Cas9 constitutively from the Rosa26 locus) and mixed them with control B cells. Primary B cells were cultured on CD40L- and BAFF-expressing feeder cells and transduced with retroviral particles expressing the sgRNAs of interest. Using this system, we have achieved complete gene knockouts in up to 92% of activated B cells.