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Title: CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells: CRISPR/Cas9-Mediated Fluorescent Tagging of Proteins in Pluripotent Stem Cells
Award ID(s):
1647837
NSF-PAR ID:
10095458
Author(s) / Creator(s):
; ; ; ; ; ; ;
Date Published:
Journal Name:
Current Protocols in Human Genetics
Volume:
96
Issue:
1
ISSN:
1934-8266
Page Range / eLocation ID:
21.11.1 to 21.11.20
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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  1. Abstract

    The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells  2019;37:65–76

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    Inherited retinal degeneration is a term used to describe heritable disorders that result from the death of light sensing photoreceptor cells. Although we and others believe that it will be possible to use gene therapy to halt disease progression early in its course, photoreceptor cell replacement will likely be required for patients who have already lost their sight. While advances in autologous photoreceptor cell manufacturing have been encouraging, development of technologies capable of efficiently delivering genome editing reagents to stem cells using current good manufacturing practices (cGMP) are needed. Gene editing reagents were delivered to induced pluripotent stem cells (iPSCs) using a Zephyr microfluidic transfection platform (CellFE). CRISPR-mediated cutting was quantified using an endonuclease assay. CRISPR correction was confirmed via digital PCR and Sanger sequencing. The resulting corrected cells were also karyotyped and differentiated into retinal organoids. We describe use of a novel microfluidic transfection platform to correct, via CRISPR-mediated homology-dependent repair (HDR), a disease-causing NR2E3 mutation in patient-derived iPSCs using cGMP compatible reagents and approaches. We show that the resulting cell lines have a corrected genotype, exhibit no off-target cutting, retain pluripotency and a normal karyotype and can be differentiated into retinal tissue suitable for transplantation. The ability to codeliver CRISPR/Cas9 and HDR templates to patient-derived iPSCs without using proprietary transfection reagents will streamline manufacturing protocols, increase the safety of resulting cell therapies, and greatly reduce the regulatory burden of clinical trials.

     
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  2. Abstract

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"CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells: CRISPR/Cas9-Mediated Fluorescent Tagging of Proteins in Pluripotent Stem Cells". <em>Current Protocols in Human Genetics</em> 96 (1). 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