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Jie Cheng, Xiaoli Chang, Yukan Duan, Cynthia Berlinicke, Donald J Zack; Probing the Differentiation of Retinal Ganglion Cells from Human Embryonic Stem Cells with Inducible CRISPR Interference. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3794.
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© ARVO (1962-2015); The Authors (2016-present)
Human embryonic stem cell (hESC)-derived retinal ganglion cells (hRGCs) are valuable in facilitating developmental studies, retinal disease modeling, and drug discovery efforts. However, the processes of how hESCs differentiate into hRGCs are not fully characterized. To understand the role of transcription and other factors in in vitro RGC differentiation, we utilized an Inducible CRISPR interference (CRISPRi) system to suppress their expression during differentiation.
Based upon our previously described H9-BRX BRN3B/TdTomato hESC reporter line, we generated an inducible CRISPRi system consisting of an inducible dCas9-KRAB and a continuously expressed sgRNA (CRISPRi-hESC). We have used the system to target the endogenous ATOH7 and SOX11 genes.
We generated H9-BRX cells with inducible expression of dCas9-KRAB, a catalytically dead Cas9 (dCas9) fused to the transcriptional repressor Krüppel-associated box (KRAB), under control of the Tet Response Element (TRE). Exogenously supplied doxycycline (dox) binds to the reverse tetracycline-controlled transactivator (rtTA) and drives expression of dCas9-KRAB by binding to its TRE promoter. We then infected these cells with lentiviral vectors containing sgRNAs complementary to a region proximal to the transcription start site (TSS) of the ATOH7 or SOX11 genes, under the control of a U6 promoter, and performed puromycin selection. We thus established cell lines ATOH7-CRISPRi-hESC and SOX11-CRISPRi-hESC. Addition of dox to stem cell culture media led to suppression of endogenous ATOH7 or SOX11 gene expression during differentiation. In the absence of dox, ATOH7-CRISPRi-hESCs and SOX11-CRISPRi-hESCs differentiate efficiently into hRGCs which can be visualized by the expression of the tdTomato reporter under control of the RGC marker gene BRN3B. While adding dox to CTRL-CRISPRi-hESCs starting on day 6 of differentiation had no effect on its RGC differentiation, adding dox to ATOH7-CRISPRi-hESCs and SOX11-CRISPRi- hESCs greatly reduced the generation of tdTomato+ RGCs.
These results confirm the importance of ATOH7 and SOX11 in the in vitro differentiation of human embryonic stem cells into RGCs and provide a system to interrogate the gene regulatory networks governing RGC differentiation as well as the role of disease genes that affects human RGC development.
This is a 2020 ARVO Annual Meeting abstract.
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