Immunotherapies targeting checkpoint inhibition and cell therapies are considered breakthroughs for cancer therapy. However, only a part of patients benefit from these treatments and resistance has been observed. Combining both approaches can potentially further enhance their efficacy. With the advent of gene editing techniques, such as clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), the elimination of checkpoint molecules became available as an option in good manufacturing practice conditions to improve persistence and efficacy. However, no data of CRISPR-Cas9 application have been reported in cord blood (CB)-derived immune cells, potentially usable for allogeneic cell therapy purposes. In this article, we describe the optimization of a protocol to deplete checkpoint molecules at the genomic level using CRISPR-Cas9 technology from CB-dendritic cells (DCs) and CB-CD8+ T cells. The protocol is based on the electroporation of a ribonucleoprotein complex, easily translatable to clinical settings. In both cell types, the knock-out (KO) was successful and did not affect cell viability. CB-DCs showed a decrease in expression of the targeted protein ranging from 50% to 95%, while CB-CD8+ T cells showed a reduction in the range of 25-45%. The procedure did not affect the stimulatory function of the CB-DCs or the response of CB-CD8+ T cells (proliferation or TNF-α production). In conclusion, we optimized a protocol to eliminate checkpoint molecules from CB-derived DCs and CD8+ T cells, with the aim to further implement allogeneic cell therapies for cancer.