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Background CD19-targeted Chimeric Antigen Receptor T-cell (CAR-T) therapies, particularly those using the FMC63-based CAR construct, have shown remarkable efficacy in treating B-cell malignancies. However, one of the prominent adverse effects of this therapy is B-cell aplasia, a condition characterized by prolonged depletion of normal B cells due to the targeting of CD19, which is expressed on both malignant and healthy B cells. B-cell aplasia can lead to hypogammaglobulinemia, increasing susceptibility to infections and necessitating immunoglobulin replacement therapy, which can impact patients’ quality of life and increase healthcare costs.Methods We introduced specific alterations in the CD19 gene to reduce CAR-T recognition while preserving CD19’s core structural and functional roles in B-cell biology. Functional assays confirmed that these engineered B cells retain normal activity and immune response capabilities while demonstrating significantly reduced sensitivity to CD19-CAR-T cell cytotoxicity in vitro and In vivo. Further we extended our study precision genome editing in hematopoietic stem cells, potential of HSPC’s transplantation as a valuable strategy to sustain CAR-T therapy’s therapeutic effects, offering a promising approach for enhancing treatment durability and patient survival in aggressive B-cell cancers. Despite the engraftment of new hematopoietic stem cells, some patients continue to experience prolonged B-cell aplasia, leading to hypogammaglobulinemia and increased infection susceptibility.Results Our analysis suggests that the persistence of CAR-T cells in circulation and the expression of CD19 on engrafted B cells contribute to this prolonged effect. CD19 gene locus-edited hematopoietic stem cells (HSCs) are being investigated as a novel approach to prevent prolonged B-cell aplasia while maintaining CAR-T efficacy.Conclusions In this study, we employ advanced gene editing techniques, such as CRISPR-Cas9 and prime editing, to modify HSCs with the goal of generating B cells that lack of FMC63-CAR binding motif on CD19 antigen but retain normal functionality, enabling resistance to FMC63-CD19-CAR-T cell depletion. These edited HSCs were transplanted into immunodeficient mouse models and demonstrated successful engraftment, B-cell lineage differentiation, and restored immunoglobulin levels without significant off-target effects. The persistence of functional, CD19-edited B cells suggests that this approach can effectively counteract B-cell aplasia while supporting immune reconstitution. Our findings indicate that gene-edited HSCs could offer a clinically translatable solution for CAR-T patients, potentially improving long-term immune recovery and quality of life by balancing therapeutic efficacy with immune system preservation.