The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits.
We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age.
This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.
■保守的精氨酸残基,斑马鱼tgfbi基因中的密码子117采用成簇规则间隔短回文重复序列(CRISPR)/Cas9方法进行靶向。Cas9VQR变体与两个靶特异性sgRNA一起使用以产生突变。通过T7核酸内切酶(T7EI)测定评估突变的存在,并通过Sanger测序评估突变的类型。在显微镜下研究3个月和1岁的突变斑马鱼的角膜混浊,并用苏木精-伊红对眼睛切片进行组织病理学评估,用于角膜沉积的马尾色和刚果红色污渍。
■我们在目标序列上实现了indel变异,导致p.Ser115_Arg117delinsLeu(c。347_353delinsT)通过非同源介导的F1修复。这种斑马鱼突变有可能模拟先前在人类病例中报道的两种致病突变:R124L和R124Ldel125-126。突变斑马鱼在3个月和1岁时没有显示任何角膜混浊或角膜沉积。
■这项研究产生了第一个斑马鱼模型,该模型模仿了TGFBI相关CD中的R124热点突变。然而,即使在1岁时进行的评估在组织病理学上也没有发现角膜中的任何沉积物。除了斑马鱼和人类之间的角膜结构差异外,这项研究增加了对斑马鱼中TGFBI相关CD建模的注意事项。