CD20 antigen has been used as a target of monoclonal antibodies (mAb) such as rituximab (RTX) in the therapy of B-cell malignancies for more than two decades. However, malignant B cells downregulate CD20 on their surface, resulting in mAb resistance and therapy failure. Therefore, it is crucial to investigate the CD20 regulation to enhance the efficacy of antiCD20 mAb. This project aimed to perform CRISPR/Cas9 knockout screening to identify genes whose disruption restores CD20 surface expression. To create a model mimicking the situation in patients who have developed resistance to mAb therapy, we generated RTX-resistant CD20-low B-cell line by chronic exposure to rituximab. These cells were transduced by the GeCKO lentiviral library to obtain a collection of single-gene knockouts. After 2.5-week cultivation, the top 5% of cells with the highest expression of CD20 were sorted out. Using next-generation sequencing, we identified gene knockouts responsible for CD20 upregulation. CRISPR/Cas9 screening revealed several genes whose disruption increased CD20 surface expression. CSK, encoding a negative regulator of Src kinases, as well as PTEN, a well-known tumor suppressor, were among the top hits. These two genes are involved in the B-cell receptor (BCR) pathway – an essential pathway in B cells. Interestingly, we identified four genes SSR1- 4, encoding all four subunits of the TRAP complex, an endoplasmic reticular complex involved in protein translocation across ER membrane. STT3A, encoding the catalytic subunit of oligosaccharyltransferase, was another ER-associated gene revealed by the screening. These results indicate that both BCR signalling and ER play an important role in CD20 regulation. Selected genes were validated, and the mechanism of their function is being investigated. The understanding of underlying mechanisms could provide a way for a potential enhancement of anti-CD20 mAb therapy.