Alipour Farzad, Ahmadraji Mana, Yektadoost Elham, Mohammadi Parvaneh, Baharvand Hossein, Basiri Mohsen
Department of Applied Cell Sciences, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran.
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Cell J. 2023 Oct 9;25(10):665-673. doi: 10.22074/cellj.2023.1989321.1225.
Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering disease caused by mutations in the gene which is responsible for coding type VII collagen. Investigating the pathological mechanisms and novel candidate therapies for RDEB could be fostered by new cellular models. The aim of this study was to employ CRISPR/Cas9 technology in the development of immortalized COL7A1-deficient keratinocyte cell lines intended for application as a cellular model for RDEB in studies.
In this experimental study, we used transient transfection to express -targeting guide RNA (gRNA) and Cas9 in HEK001 immortalized keratinocyte cell line followed by enrichment with fluorescent-activated cell sorting (FACS) via GFP expressing cells (GFP+ HEK001). Homogenous single-cell clones were then isolated, genotyped, and evaluated for type VII collagen expression. We performed a scratch assay to confirm the functional effect of knockout.
We achieved 46.1% (P<0.001) efficiency of in/del induction in the enriched transfected cell population. Except for 4% of single nucleotide insertions, the remaining in/dels were deletions of different sizes. Out of nine single expanded clones, two homozygous and two heterozygous -deficient cell lines were obtained with defined mutation sequences. No off-target effect was detected in the knockout cell lines. Immunostaining and western blot analysis showed lack of type VII collagen (COL7A1) protein expression in these cell lines. We also showed that -deficient cells had higher motility compared to their wild-type counterparts.
We reported the first isogenic immortalized -deficient keratinocyte lines that provide a useful cell culture model to investigate aspects of RDEB biology and potential therapeutic options.
隐性营养不良型大疱性表皮松解症（RDEB）是一种遗传性皮肤脆性疾病，最终会导致致命的水疱形成，由负责编码VII型胶原蛋白的基因突变引起。新的细胞模型有助于研究RDEB的病理机制和新型候选疗法。本研究的目的是利用CRISPR/Cas9技术开发永生化的COL7A1缺陷角质形成细胞系，用于在研究中作为RDEB的细胞模型。
在本实验研究中，我们通过瞬时转染在HEK001永生化角质形成细胞系中表达靶向COL7A1的引导RNA（gRNA）和Cas9，随后通过绿色荧光蛋白（GFP）表达细胞（GFP+HEK001）进行荧光激活细胞分选（FACS）富集。然后分离出均匀的单细胞克隆，进行基因分型，并评估VII型胶原蛋白的表达。我们进行了划痕试验以确认COL7A1敲除的功能效应。
在富集的转染细胞群体中，我们实现了46.1%（P<0.001）的插入/缺失诱导效率。除了4%的单核苷酸插入外，其余的插入/缺失均为不同大小的缺失。在九个单克隆扩增克隆中，获得了两个纯合和两个杂合COL7A1缺陷细胞系，其突变序列明确。在敲除细胞系中未检测到脱靶效应。免疫染色和蛋白质印迹分析表明，这些细胞系中缺乏VII型胶原蛋白（COL7A1）蛋白表达。我们还表明，与野生型细胞相比，COL7A1缺陷细胞具有更高的运动性。
我们报道了首个同基因的永生化COL7A1缺陷角质形成细胞系，该细胞系为研究RDEB生物学方面和潜在治疗选择提供了有用的细胞培养模型。
