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Phuong T Lam, Christian Gutierrez, Katia Del Rio-Tsonis, Michael L Robinson; A Fluorescent VSX2 Reporter for Neural Retina Differentiation Created in hiPSCs by CRISPR/Cas9. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4578.
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© ARVO (1962-2015); The Authors (2016-present)
Neural retina (NR) degeneration leads to vision loss, affecting millions of people worldwide. Therapies to arrest this degenerative process remain limited. Human induced pluripotent stem cell (hiPSC)-derived NR progenitors hold great potential for cell-based therapies aimed at NR regeneration. Expression of VSX2 transcription factor specifically marks NR progenitor (NRP) cells early in eye development before becoming restricted to mature retinal biopolar neurons. Here, we describe a CRISPR/Cas9 to insert Cyan Fluorescent Protein (CFP) reporter gene into the VSX2 locus to label living human NRP cells without disrupting the function of the endogenous VSX2 allele
Homology directed repair (HDR), following CRISPR/Cas9-mediated double strand DNA breakage in hiPSCs, facilitated the replacement of the VSX2 stop codon, in frame with viral P2A peptide fused to CFP reporter gene. This accomplishment resulted from co-electroporation a HDR DNA template with Cas9 vector encoding specific gRNA into the CB-hiPSC6.2 cell line. PCR primer sets spanning each side of HDR junctions unambiguously identified successfully targeted hiPSC from G418 resistant cells. The hiPSC-VSX2/CFP clones were differentiated to NRPs as assessed by specific expression of VSX2 and CFP (blue) fluorescence. At Day 0 (D0) and D20 of NR differentiation, cells were examined for mRNA expression of VSX2-P2A-CFP, and eye field transcription factors (Pax6, RX, LHX2, SIX3, and SIX6). D20 cells were also examined for VSX2 and CFP protein by double immunofluorescence (d-IF).
Seven of forty eight G418-resistant clones exhibited the expected PCR fragments on each side of the HDR junction as assessed by both size and sequence. One of these successfully targeted hiPSCs-VSX2/CFP clones was used to create optic cups in vitro. Although negative for both VSX2 expression and CFP fluorescence at D0, at D20 of NR differentiation optic cups exhibited VSX2-P2A-CFP, Pax6, RX, LHX2, SIX3, and SIX6 mRNA expressions as well as blue fluorescence (figure 1). VSX2 and CFP proteins were also detected in these D20 optic cups by d-IF
A CRISPR-Cas9 based genome editing strategy successfully generated a VSX2 reporter cell line in hiPSCs. These cells will facilitate a simple visualization of NR progenitor during hiPSC-NR differentiation in vitro
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Figure 1: D30 NR-derived hiPSCs under bright-field (A), real-time CFP expression (B), and both in (C).
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