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Phuong Lam, Christian Gutierrez, Katia Del Rio-Tsonis, Michael L. Robinson; CRISPR/Cas9 Edited hiPSCs to Model Neural Retina Differentiation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):573.
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© ARVO (1962-2015); The Authors (2016-present)
Early in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retina progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PhRs), respectively. The growing utility of human induced pluripotent stem cells (hiPSCs), both for modeling human retinal development and as a potential source for treating retinal diseases, makes the optimization of retina differentiation protocols extremely important. Here, we describe a CRISPR/Cas9 strategy to generate three transgenic hiPSCs lines that utilize the endogenous VSX2, BRN3b, and RCVRN promoters to specifically express fluorescent proteins in NRPs, RGCs and PhRs without disrupting the function of the endogenous alleles.
Homology directed repair (HDR) facilitated the replacement of the VSX2, BRN3b, and RCVRN stop codons, with a viral P2A peptide fused to cyan fluorescent protein (CFP), green fluorescent protein (GFP), and mCherry reporter genes, respectively. This was accomplished by co-electroporating an HDR template and sgRNA/Cas9 vectors into CB-hiPSC6.2 cells, followed by G418 selection. PCR primer sets spanning each side of the HDR junctions identified successfully targeted hiPSCs. The identified clones were differentiated into three-dimensional retinal cups (3-D RC) to verify for fluorescence expression in living RCs, mRNAs expression of eye field transcription factors, and immunofluorescence (IF) of specific retinal cell types.
14.5% (7/48), 13% (10/77), and 6% (1/18) of G418 resistant hiPSC clones exhibited the expected PCR products (sequence verified) on each side of the HDR junction for the VSX2/CFP, BRN3b/GFP, and RCVRN/mCherry targeting constructs, respectively. One successfully targeted clone from each line was used to create 3-D RCs in vitro. The 3-D RCs exhibited significant transcriptional up-regulation of eye field transcription factors (Pax6, RX, LHX2, SIX3, and SIX6). Additionally, the RCs exhibited CFP expression starting at 30 days (D30), GFP on D35, and mCherry on D48, marking the real-time expression of VSX2, BRN3b and RCVRN, respectively. Ninety days after establishing 3-D RCs, double IF confirmed that NRPs co-expressed both VSX2 and CFP, RGCs co-expressed both BRN3b and GFP, and PhRs co-expressed RCVRN and mCherry.
CRISPR-Cas9 facilitated the generation of three independent hiPSC lines that facilitate a simple visualization of NRP, RG, PhR during hiPSC to NR differentiation in vitro.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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