Expression data in PAX6 depleted corneal epithelial cells. Expression data in PAX6 depleted corneal epithelial cells

PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates thecell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify thefunction of PAX6 in human CECs using gene knockout via the clustered regularly interspaced shortpalindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAsfor different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but becamelarger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specificand included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1(ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes wereprimarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). Thesefindings suggest that PAX6 maintains CEC identity by regulating differentiation. Overall design: Human primary corneal epithelial cells depleted PAX6 with CRISPR/Cas9

PAX6作为一种配对盒转录因子（paired box transcription factor），对眼球发育至关重要。然而，其调控人角膜上皮细胞（corneal epithelial cells, CECs）细胞身份的分子机制尚未完全阐明。
本研究旨在借助成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）及其关联蛋白9（CRISPR associated protein 9, Cas9）系统开展基因敲除实验，以明确PAX6在人角膜上皮细胞中的功能。我们针对PAX6的不同靶点设计了向导RNA（guide RNAs）。
PAX6敲除的人角膜上皮细胞仍维持上皮细胞形态，但细胞体积增大。通过微阵列进行的全局转录组分析显示，下调基因主要为角膜上皮细胞特异性基因，包括角蛋白12（keratin 12）、角蛋白3（keratin 3）、簇蛋白（clusterin, CLU）、醛脱氢酶3家族A1成员（aldehyde dehydrogenase 3 family member A1, ALDH3A1）、血管生成素样7（angiopoietin-like 7, ANGPTL7）及转酮醇酶（transketolase, TKT）；而上调基因主要与表皮分化相关，包括角蛋白10（keratin 10）、角蛋白1（keratin 1）、兜甲蛋白（involucrin, IVL）及丝聚蛋白（filaggrin, FLG）。
上述研究结果表明，PAX6通过调控分化过程以维持人角膜上皮细胞的细胞身份。
整体实验设计：采用CRISPR/Cas9系统敲除人原代角膜上皮细胞中的PAX6基因。