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Koji Kitazawa, Takafusa Hikichi, Takahiro Nakamura, Chie Sotozono, Shigeru Kinoshita, Shinji Masui; Loss of human corneal epithelial cell identity exhibited by deletion of PAX6 using the CRISPR/Cas9 system. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
The findings of numerous animal studies have demonstrated that the paired box 6 (PAX6) gene plays an important role during ocular surface development. The purpose of this present study was to clarify how PAX6 regulates the cell identity of adult human corneal epithelial cells (HCECs).
To perform a functional analysis of PAX6 via gene knockout, we used lentiCRISPR, a lentivirus vector carrying both guide RNA targeted for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). We designed guide RNA for the different targets in PAX6 using a web tool, which provided data on all off-target sites for the predicted target. After lentiviral transduction followed by selection with 1 mg/ml puromycin, primary HCECs were harvested at day 7 and then assessed by western blotting, microarray, and quantitative real-time polymerase chain reaction. An empty vector was used as a control.
Western blot analysis found that abolished PAX6 protein in CECs transduced two different lentiCRISPRs with single-guide RNA for PAX6 compared to the control. PAX6-deleted CECs exhibited no apparent change in terms of morphology compared to the control, and the cell size became larger and took a longer period of time to reach confluence. Global analyses between CECs and PAX6-deleted CECs using microarray revealed that 760 genes were differentially expressed by more than two fold between PAX6-deleted CECs and CECs, with 371 genes upregulated and 389 genes downregulated. The downregulated genes were enriched to CEC-specific genes including keratin (K)12, K3, clusterin, lumican, aldehydedehydrogenase3family, memberA1 (ALDH3A1), and angiopoietin-like 7 (ANGPTL7). The upregulated genes were enriched to epidermis-related genes such as K10, K1, and involucrin.
The findings of this study show that PAX6-regulated genes related to differentiation, transparency, and neovascularization in the cornea inhibited abnormal differentiation and determined the cell identity of adult HCECs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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