Knockdown of HMGB1 significantly reduces V(D)J recombination in vivo. (A) Schematic of the steps used in CRISPR/Cas9-mediated knockdown of HMGB1. Transduction with lentiviruses and antibiotic selection steps are shown. (B) Representative western blots showing the levels of HMGB1 and full-length (fl) and C-terminal deletion (ΔC) HMGB2 relative to a β-tubulin control. Antibodies that specifically recognize HMGB2ΔC are unavailable, as the epitope lies at the C terminus. However, the core regions of HMGB1 and HMGB2 are 85.5% identical, and the anti-HMGB1 antibody cross-reacts with HMGB2 (indicated by the asterisk). (C) V(D)J recombination levels with the RAG1 proteins shown in WT NIH3T3 cells (blue bars), when the KRAB repressor as introduced in the absence of guide RNAs (purple bars), when HMGB1 has been knocked down (red bars), or when HMGB1 (dark green bars) or HMGB2 full-length (light green bars) or HMGB2ΔC (turquoise bars) have been reintroduced. n = 3; error bars show SEM. The changes in recombination activity are statistically significant. Student t test; **P = .01; ***P = .001. The endogenous HMGB2 in NIH3T3 cells could account for residual recombination activity after HMGB1 knockdown. ns, nonspecific.