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Cell Journal، جلد ۲۵، شماره ۱۰، صفحات ۶۶۵-۶۷۳
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عنوان انگلیسی CRISPR/Cas9-Mediated Generation of COL7A1-Deficient Keratinocyte Model of Recessive Dystrophic Epidermolysis Bullosa
چکیده انگلیسی مقاله Objective: Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin fragility and ultimately lethal blistering
disease caused by mutations in the COL7A1 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 ex vivo studies.
Materials and Methods: In this experimental study, we used transient transfection to express COL7A1-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 COL7A1 knockout.
Results: 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 COL7A1-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
COL7A1-deficient cells had higher motility compared to their wild-type counterparts.
Conclusion: We reported the first isogenic immortalized COL7A1-deficient keratinocyte lines that provide a useful cell
culture model to investigate aspects of RDEB biology and potential therapeutic options.
کلیدواژه‌های انگلیسی مقاله COL7A1, CRISPR/CAS9, Keratinocyte, Recessive dystrophic epidermolysis bullosa
نویسندگان مقاله Farzad Alipour |
Department of Applied Cell Sciences, Faculty of Basic Sciences and Advanced Medical Technologies, Royan Institute, ACECR, Tehran, Iran

Mana Ahmadraji |
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Elham Yektadoost |
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Parvaneh Mohammadi |
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Hossein Baharvand |
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

Mohsen Basiri |
Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran

نشانی اینترنتی https://www.celljournal.org/article_706590_77f773b6a23e7b97c477335c66ef00cc.pdf
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