Widespread cryptic RAG-mediated recombination in developing T lymphocytes

V(D)J recombination generates antigen receptor diversity through the targeted activity of the RAG1/2 recombinase, but the extent to which RAG also engages cryptic genomic targets during normal lymphocyte development remains poorly defined. Here, we developed a targeted PCR-sequencing approach to detect and quantify rare RAG-mediated rearrangements in mouse thymocytes. We first examined the TCRβ locus and found that four of its twelve pseudogenes undergo detectable rearrangement in vivo, despite being considered non-functional components of the repertoire. Extending this analysis across the locus revealed 33 previously uncharacterized cryptic recombination sites involved in non-functional rearrangements with DJβ segments. These events occurred across both the Vβ region and the largely inaccessible Vβ30–Dβ1 intergenic region, with individual cryptic sites spanning a broad range of recombination frequencies. Cryptic sites were highly heterogeneous in sequence and chromatin context: neither RSS resemblance, predicted Z-DNA formation, local epigenetic features, nor chromosomal interactions reliably distinguished sites with detectable recombination from those at which recombination was not detected. We further identified additional cryptic RAG-mediated rearrangements at the Bcl11b locus, demonstrating that such events are not restricted to antigen receptor loci. Together, these findings reveal an unexpectedly broad landscape of low-frequency RAG-mediated DNA rearrangement in developing T lymphocytes and suggest that cryptic target selection cannot be explained solely by the local genetic and epigenetic features examined here.