Knock‐in strategy at 3′‐end of Crx gene by CRISPR/Cas9 system shows the gene expression profiles during human photoreceptor differentiation

Abstract

Fluorescent reporter gene knock-in induced pluripotent stem cell (iPSC) lines have been used to evaluate the efficiency of differentiation into specific cell lineages. Here, we report a knockin strategy for the generation of human iPSC reporter lines in which a 2A peptide sequence and a red fluorescent protein (E2-Crimson) gene were inserted at the termination codon of the cone-rod homeobox (Crx) gene, a photoreceptor-specific transcriptional factor gene. The knock-in iPSC lines were differentiated into fluorescence-expressing cells in 3D retinal differentiation culture, and the fluorescent cells also expressed Crx specifically in the nucleus. We found that the fluorescence intensity was positively correlated with the expression levels of Crx mRNA and that fluorescent cells expressed rod photoreceptor-specific genes in the later stage of differentiation. Finally, we treated the fluorescent cells with DAPT, a Notch inhibitor, and found that DAPT-enhanced retinal differentiation was associated with up-regulation of Crx, Otx2 and NeuroD1, and down-regulation of Hes5 and Ngn2. These suggest that this knock-in strategy at the 3 0 -end of the target gene, combined with the 2A peptide linked to fluorescent proteins, offers a useful tool for labeling specific cell lineages or monitoring expression of any marker genes without affecting the function of the target gene.

Taurine rescues mitochondria-related metabolic impairments in the patient-derived induced pluripotent stem cells and epithelial-mesenchymal transition in the retinal pigment epithelium

“…MELAS patient-derived (A24#1–3) and control human iPSCs (454E2, RIKEN BRC, Japan) [ 38 ] were cultured in a feeder-free condition as described previously [ 27 ]. Cells were maintained in Nutristem (Riprocell, Japan) on Matrigel (human embryonic stem cell-qualified Matrigel, BD, NJ, USA) coated 6-well plates.…”

“…Here, we generated MELAS patient-derived iPSC lines harboring the mtDNA A3243G mutation and expressing high levels of the mutant mtDNA. We documented metabolic changes in MELAS iPSCs, demonstrated the phenotype of the RPE differentiated from these iPSCs [ [25] , [26] , [27] , [28] , [29] ], and analyzed the protective effects of taurine. The undifferentiated pluripotent stem cells (PSCs) possess functional OXPHOS machinery; however, it is decoupled from glycolysis, which is the major energy source in PSCs, similarly to the situation in cancer cells [ [30] , [31] , [32] ].…”

Guiding Lights in Genome Editing for Inherited Retinal Disorders: Implications for Gene and Cell Therapy

“…On a different note, CRISPR/Cas technology in iPSCs has been used for fluorescent reporter gene knock-in at the termination codon of the cone-rod homeobox ( Crx ) gene, a photoreceptor-specific transcription factor gene. This allows the real-time monitoring of photoreceptor differentiation [ 158 ], demonstrating the interest of this technology also for fundamental research.…”

Microfluidic processing of stem cells for autologous cell replacement

“…For instance, we and others have shown that following subretinal transplantation iPSC‐derived photoreceptor precursors cells are able to restore retinal function in animal models of retinal degenerative blindness 1‐10 . Following decades of development, the vast majority of the differentiation protocols reported faithfully recapitulate normal retinal development, which means that they give rise to each of the different cell types found in the retina 10‐40 . For therapeutic photoreceptor cell replacement, enrichment of photoreceptor precursor cells away from the unwanted cell types (ie, retinal ganglion cells, bipolar inter neurons, retinal pigmented epithelial [RPE] cells, etc) will be desirable.…”

“… 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 Following decades of development, the vast majority of the differentiation protocols reported faithfully recapitulate normal retinal development, which means that they give rise to each of the different cell types found in the retina. 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 For therapeutic photoreceptor cell replacement, enrichment of photoreceptor precursor cells away from the unwanted cell types (ie, retinal ganglion cells, bipolar inter neurons, retinal pigmented epithelial [RPE] cells, etc) will be desirable.…”