Chimeric antigen receptor (CAR) T cell therapy is a promising approach to treat cancer, such as B-cell malignancy [1]. However, the current standard treatment requires autologous adoptive cell transfer, which is expensive and time-consuming. For newborn and elder patients, it is often difficult to obtain enough T cells with good quality to generate patient-specific CAR-T cells. To make CAR-T therapy more accessible, it is highly desirable to develop an allogeneic adoptive transfer strategy, in which universal CAR-T cells derived from T cells from healthy donors can be applied to treat multiple patients. For this strategy to work, the αβ T-cell receptor (TCR) on allogeneic CAR-T cells needs to be eliminated to avoid graft-versus-host-disease (GVHD), and human leukocyte antigens class I (HLA-Is) on CAR-T cells need to be removed to minimize their immunogenicity. Previous studies have shown that mutation in TCRα subunit constant (TRAC) leads to loss of αβ TCR on T-cell surface [2], and beta-2 microglobulin (B2M) is essential for cell-surface expression of HLA-I heterodimers [3]. Thus, we attempted to target TRAC and B2M genes in CAR-T cells. Considering blocking programmed death-1 (PD-1) signaling can effectively treat cancers via reversing immunosuppression, we also targeted PD-1 in CAR-T cells to render them nonresponsive to PD-1 signaling [4]. To generate universal and more potent CAR-T cells described above, multiple genes need to be eliminated simultaneously. While Torikai et al.[2, 5] have used ZFN to knockout TRAC [2] and HLA-A [5] individually in CAR-T cells, they did not test the function of these edited cells in vivo. Although researchers from Cellectis have used TALEN to simultaneously target TRAC and CD52 or deoxycytidine kinase in CD19 CAR-T cells [6, 7] and demonstrated the anti-tumor activity of the edited CAR-T cells in a lymphoma murine model [6], it remains to be tested whether CAR-T cells lacking B2M can still function properly. More importantly, whether clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system (CRISPR-Cas9)[8] can be applied to perform multiplex gene editing in CAR-T cells has not been evaluated. Previously, we have demonstrated that up to five genes can be disrupted simultaneously in mouse embryonic stem cells with high efficiency using CRISPR-Cas9 [9]. In this study, we developed protocols to efficiently generate CAR-T cells with two (TRAC and B2M) or three genes (TRAC, B2M, and PD-1) disrupted and tested their an-