Abstract Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils. X91+-CGD mutations lead to a loss of function but with a normal expression of mutated NOX2 proteins. It is therefore necessary to ensure that these mutations are indeed responsible for the loss of activity for genetic counseling. Thus to ascertain the identity of the neutrophil-like model used to study the functional impact of X91+-CGD mutations, we developed a new X-CGD cellular model using the clustered regularly interspaced short palindromic repeats associated Cas9 protein (CRISPR-Cas9) technology (all-in-one Cas9 SmartNucleaseTM Plasmid Expression System) in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Graphical Abstract