Background <p>Antibodies are essential mediators of adaptive immunity, providing defense against pathogens and serving as critical tools in therapeutics of infection and cancer. Antibody diversification, including class switch recombination (CSR), is tightly regulated by ubiquitination, a post-translational modification, that modulates protein stability and functional activity.</p> Methods and results <p>Using CRISPR/Cas9 screening in B cells, we investigated the role of ubiquitin-related enzymes in CSR. Validation experiments with LentiCRISPR-sgAID stable cell lines, which showed significant inhibition of CSR, confirmed that it works in the CSR model CH12F3 cell. Screening of 35 E2 ubiquitin-conjugating enzymes and 85 deubiquitinases (DUBs) identified Ube2v1, an E2 enzyme, as a potent suppressor of CSR. Specifically knockdown of Ube2v1 significantly enhanced CSR efficiency, whereas its overexpression inhibited CSR without affecting germline transcripts (GLTs) or activation-induced cytidine deaminase (AID). Intriguingly, although Ube2v1 canonically functions with Ube2n to mediate polyubiquitination, overexpression and knockdown of Ube2n had no detectable effect on CSR.</p> Conclusions <p>Our CRISPR screening identified multiple components of ubiquitin pathway that regulate CSR and established Ube2v1 as a novel inhibitor. Ube2v1 functions independently of expression of GLTs and AID as well as its canonical partner Ube2n, revealing a non-canonical role. These findings underscore the complexity of post-translational regulation of humoral immunity and suggest Ube2v1 as a potential therapeutic target for modulating antibody responses.</p>