High-throughput sequencing of Epidermolysis Bullosa- and Epidermolytic Ichthyosis-associated genomic regions in primary keratinocytes (Ion Torrent amplicon panel)

We developed a comprehensive AmpliSeq panel for NGS analysis of EB and EI-asociated genomic loci. These included mutation hotspots and predicted off-target sites for gene editing agents. Some of the data generated was used to analyse the off-target and on-target capabilities of a KRT10-specific TALEN (6.21) in the development of a gene editing therapeutic option for Epidermolytic Ichthyosis. In this study, the KRT10 on-target 17q21.2 (KRT10 exon 6) and 8 in silico predicted off-targets 4q12 (non-coding), 6q21 (non-coding), 6p22.1 (non-coding), 4q31.3 (FHDC1 intron 6), 1p21 (non-coding), 2p25 (non-coding), 5q15 (non-coding), 1p36.3 (non-coding) were analysed. Other genomic loci were included on the amplicon panel. Although these were not analysed in this study, they are included in the provided metadata. NGS data was assessed in primary patient keratinocytes (Prim_EI_Unmodified) and TALEN-treated primary patient keratinocytes (Prim_EI_T6.21). Other samples were included on the amplicon panel, but are not in the provided metadata. Despite high on-target activity, TALEN 6.21 activity was not observed at off-target sites.

我们开发了一款覆盖全面的AmpliSeq扩增子测序panel（AmpliSeq panel），用于针对大疱性表皮松解症（Epidermolysis Bullosa, EB）与表皮松解性鱼鳞病（Epidermolytic Ichthyosis, EI）相关基因组位点的下一代测序（Next-Generation Sequencing, NGS）分析。该panel涵盖了基因编辑工具的突变热点区域与预测脱靶位点。本研究生成的部分数据，被用于分析靶向角蛋白10（Keratin 10, KRT10）的转录激活因子样效应物核酸酶（Transcription Activator-Like Effector Nuclease, TALEN）(6.21)在开发表皮松解性鱼鳞病基因编辑治疗方案中的脱靶与靶向编辑能力。本研究中，我们对靶向KRT10的靶向位点17q21.2（位于KRT10第6外显子）以及8个计算机预测的脱靶位点（4q12（非编码区）、6q21（非编码区）、6p22.1（非编码区）、4q31.3（FHDC1基因第6内含子）、1p21（非编码区）、2p25（非编码区）、5q15（非编码区）、1p36.3（非编码区））进行了分析。该AmpliSeq panel还涵盖了其他基因组位点。尽管本研究未对这些位点进行分析，但它们已包含在本次提供的元数据中。我们对患者原代未修饰角质形成细胞（Prim_EI_Unmodified）以及经TALEN 6.21处理的患者原代角质形成细胞（Prim_EI_T6.21）的NGS数据进行了评估。该扩增子测序panel还纳入了其他样本，但这些样本未包含在本次提供的元数据中。尽管该TALEN 6.21具有较高的靶向编辑活性，但在所有脱靶位点均未检测到其编辑活性。