Modeling Human Severe Combined Immunodeficiency and Correction by CRISPR/Cas9-Enhanced Gene Targeting.
Authors:
Chia-Wei Chang, Yi-Shin Lai, Erik Westin, Alireza Khodadadi-Jamayran, Kevin M. Pawlik, Lawrence S. Lamb Jr., Frederick D. Goldman, Tim M. Townes
In:
Source:
Cell Rep
Publication Date:
(
2015
)
Issue:
12(10)
:
1668-77
Experiment
IPSCs were treated with 0.25% trypsin for 5 min to generate single-cell suspensions. After washing twice with 1 × PBS, 1–2 million cells were mixed with 5 µg of JAK3 repair plasmid and 5 µg of either pX330-JAK3 or pX335-JAK3 plasmid for Nucleofection (Human Stem Cell Nucleofector Kit, program A-023, Lonza) and subsequent plating onto MEFs. Two to 4 days later, hES medium containing 30 µg/ml of G418 was added to the plates to select for drug-resistant colonies. Lonza summary: The paper describe a methode to study the genetics of human lymphopoiesis by gene disruption in iPSC follwoed by differentiation, using Nucleofction and CRISPR. It shows one way to go for gene therapy of human immunodeficiencies.
Abstract
Mutations of the Janus family kinase JAK3 gene cause severe combined immunodeficiency (SCID). JAK3 deficiency in humans is characterized by the absence of circulating T cells and natural killer (NK) cells with normal numbers of poorly functioning B cells (T(-)B(+)NK(-)). Using SCID patient-specific induced pluripotent stem cells (iPSCs) and a T cell in vitro differentiation system, we demonstrate a complete block in early T cell development of JAK3-deficient cells. Correction of the JAK3 mutation by CRISPR/Cas9-enhanced gene targeting restores normal T cell development, including the production of mature T cell populations with a broad T cell receptor (TCR) repertoire. Whole-genome sequencing of corrected cells demonstrates no CRISPR/Cas9 off-target modifications. These studies describe an approach for the study of human lymphopoiesis and provide a foundation for gene correction therapy in humans with immunodeficiencies.
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