Targeted Repair of CYBB in X-CGD iPSCs Requires Retention of Intronic Sequences for Expression and Functional Correction.
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91phoxprotein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phoxexpression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91phoxexpression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91phoxand ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA. Electronic address: sweeneyco@nih.gov.
Colin L
CL
Sweeney
Center for Molecular Medicine, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA.
Jizhong
J
Zou
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Uimook
U
Choi
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Randall K
RK
Merling
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Alexander
A
Liu
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Aaron
A
Bodansky
Molecular Cytogenetics Section, MCGP, Center for Cancer Research, National Cancer Institute, NIH, Frederick, MD 21702, USA.
Sandra
S
Burkett
Department of Life Science, Chung-Ang University, Seoul 06974, Korea.
Jung-Woong
JW
Kim
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Suk See
SS
De Ravin
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Harry L
HL
Malech
D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D005091:Exons; D000072669:Gene Editing; D005786:Gene Expression Regulation; D023061:Gene Order; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D005822:Genetic Vectors; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000378:metabolism* / Q000628:therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D007438:Introns; D008562:Membrane Glycoproteins / Q000235:genetics*; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics*; D052416:Targeted Gene Repair; D019076:Transgenes
D008562:Membrane Glycoproteins; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases
CRISPR; TALEN; X-linked chronic granulomatous disease; iPSC; intron; targeted correction