CRISPR Screen to Identify Novel Secreted Epidermal Regulators

项目摘要 表皮由不同的细胞层组成，这些细胞层协同工作以执行各种各样的基本功能。 功能协调发展的角质形成细胞包括表皮的大部分细胞，并且形成与表皮不同的层。 基底增殖层至最外层皮质层。所有层的角质形成细胞分泌多种 在表皮的正常功能中具有关键作用的蛋白质因子，包括结构蛋白， 抗微生物肽、蛋白酶和蛋白酶抑制剂以及细胞-细胞信号分子。尽管 细胞外蛋白质在表皮稳态中的公认作用，对这些蛋白质的综合评价 分泌的因子-角质形成细胞“分泌物组”-还没有进行。 这个提议描述了我们已经鉴定的分泌性表皮蛋白的综合目录 通过质谱分析。对祖细胞和分化的角质形成细胞的分泌组的分析揭示了 >100种在表皮中功能未知的新因子。一个接一个地研究它们， 候选办法将是一项时间和资源密集型工作。 在这里，我们建议使用基于CRISPR/Cas9的遗传筛选来系统地鉴定功能性的 角质形成细胞分泌影响表皮更新和分化的蛋白质。这项努力将 加速我们对皮肤分泌蛋白功能的理解，我们为 确定新的皮肤生物标志物和治疗方法。 1

PROJECT SUMMARY The epidermis consists of distinct cell layers that work in concert to execute a wide variety of essential functions. Keratinocytes comprise the majority of the cells of the epidermis, and form distinct strata from the basal proliferative layer to the outermost cornified layer. Keratinocytes of all strata secrete a variety of protein factors that have critical roles in the proper function of the epidermis, including structural proteins, antimicrobial peptides, proteases and protease inhibitors, and cell-cell signaling molecules. Despite the recognized role of extracellular proteins in epidermal homeostasis, a comprehensive evaluation of these secreted factors—the keratinocyte “secretome”— has not been performed. This proposal describes the comprehensive catalog of secreted epidermal proteins we have identified by mass spectrometry. Analysis of the secretome of progenitor and differentiated keratinocytes reveals >100 novel factors whose function in the epidermis is unknown. Studying them one-by-one or through a candidate approach would be a time and resource-intensive effort. Here, we propose to use a CRISPR/Cas9-based genetic screen to systematically identify the functional keratinocyte secreted proteins that impact epidermal renewal and differentiation. This effort would accelerate our understanding of the function of secreted proteins in the skin, and we pave the way to identify new skin biomarkers and therapeutics. 1