title: CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants.
abstract: Chronic granulomatous disease (CGD) is characterized by defects in the production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for the treatment of CGD in human hematopoietic cells requires preclinical transplant models. The use of the lymphocyte-deficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized for targeted knockout of mouse Cybb on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting Cybb exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at least one Cybb allele based on sequence analysis of tail snip DNA. A female mouse heterozygous for a 235-bp deletion in Cybb exon 1 was bred to an NSG male to establish the X-CGD NSG mouse strain, NSG.Cybb[KO]. Resulting male offspring with the 235 bp deletion were found to be defective for production of ROS by neutrophils and other phagocytes, and demonstrated increased susceptibility to spontaneous bacterial and fungal infections with granulomatous inflammation. The establishment of the phagocyte-defective NSG.Cybb[KO] mouse model enables the in vivo assessment of gene and cell therapy strategies for treating CGD in human hematopoietic cell transplants without obfuscation by functional mouse phagocytes, and may also be useful for modeling other phagocyte disorders in humanized NSG mouse xenografts.
authors.affiliation: 1 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases , National Institutes of Health, Bethesda, Maryland.
authors.forename: Colin L
authors.initials: CL
authors.lastname: Sweeney
authors.affiliation: 1 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases , National Institutes of Health, Bethesda, Maryland.
authors.forename: Uimook
authors.initials: U
authors.lastname: Choi
authors.affiliation: 2 Transgenic Core, National Heart, Lung, and Blood Institute, National Institutes of Health , Bethesda, Maryland.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Liu
authors.affiliation: 1 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases , National Institutes of Health, Bethesda, Maryland.
authors.forename: Sherry
authors.initials: S
authors.lastname: Koontz
authors.affiliation: 3 Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke , National Institutes of Health, Bethesda, Maryland.
authors.forename: Seung-Kwon
authors.initials: SK
authors.lastname: Ha
authors.affiliation: 1 Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases , National Institutes of Health, Bethesda, Maryland.
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
mesh_terms: D000818:Animals; D001483:Base Sequence; D019070:Cell Lineage; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D015316:Genetic Therapy; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000628:therapy*; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation / Q000235:genetics; D000074662:NADPH Oxidase 2 / Q000235:genetics*; D010641:Phenotype; D017382:Reactive Oxygen Species / Q000378:metabolism; D033581:Stem Cell Transplantation; D014161:Transduction, Genetic
chemical_list: D017382:Reactive Oxygen Species; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2
keywords: CRISPR/Cas9; Cybb; NSG mouse; chronic granulomatous disease; knockout