We generated helper-dependent HDAd5/35⁺⁺ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34⁺ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35⁺⁺ vector that expressed two anti-CRISPR (Acr) peptides (AcrII4 and AcrII2) capable of binding to the CRISPR/Cas9 complex (HDAd-Acr). CD34⁺ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34⁺ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.