Linear RAG scanning mediates editing of Igκ variable region repertoires

. Here we describe studies that elucidate the physiologically critical secondary Igk recombination mechanism. Primary deletional and inversional VκJκ1 joins, respectively, delete or displace Cer/Sis, creating a pre-B cell population that harbours secondary VκJκ1-based RCs across the Vκ locus and leaves most unrearranged Vκs immediately upstream of secondary RCs in deletional orientation. High-throughput assays demonstrated that RAG scanning from secondary VκJκ1-based RCs, collectively, extends linearly across the Vκ locus in primary pre-B cell populations. Correspondingly, studies of induced pluripotent stem (iPS) cell-generated mouse models or cell lines with physiological VκJκ-rearrangements further revealed that deletional and, originally, inversional Vκs are mostly captured by Jκ2-5-based secondary RCs in deletional orientation by means of linear RAG scanning. Strong Vκ-RSSs contribute to restricting secondary rearrangements, including potential editing rearrangements, to Vκs immediately upstream of a given secondary RC and support, at a lower level, linear scanning-based inversional Vκ-to-Jκ rearrangements. Our findings implicate Cer/Sis deletion and/or displacement as a developmental switch that converts the two-loop-based diffusional primary Igk rearrangement mechanism into a one-loop-based linear scanning secondary rearrangement mechanism.