Optimized immunoglobulin knock-ins using Cas9 reveal peritoneal B cell lineage relationships in vivo

Immunoglobulin (Ig) knock-in mice are valuable tools in basic and translational immunological research. Here we present "Speed-Ig," a rapid Cas9-based method for generating Ig knock-in mouse lines with high on-target integration rates at both heavy and light chain alleles. With standardized target sites and promoter regions, Speed-Ig mice can be used for comparative studies of B cell biology and vaccine optimization in vivo. We used Speed-Ig to create panels of mice with Ig pairs derived from B-1a, B-1b, and B-2 cells. Surprisingly, B-1b and B-2 Ig pairs drove both B-1b and B-2 phenotypes, suggesting a previously unknown lineage relationship between these subsets. We then confirmed the B-1:B-2 relationship with transcription factor reporter lines and through adoptive cell transfer experiments. In summary, our Ig knock-in approach facilitated the discovery of previously unappreciated aspect of innate-like B cell biology.