Division of Biomedical Engineering, National Defense Medical College Research Institute, Tokorozawa, Saitama, 359-8513, Japan.
Animal Genome Unit, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, 305-0901, Japan.
IUBMB Life. 2019 Jul;71(7):835-844. doi: 10.1002/iub.2004. Epub 2019 Jan 11.
Genome editing, as exemplified by CRISPR/Cas9, is now recognized as a powerful tool for the engineering of endogenous target genes. It employs only two components, namely, Cas9 in the form of DNA, mRNA, or protein; and guide RNA (gRNA), which is specific to a target gene. When these components are transferred to cells, they create insertion/deletion mutations (indels) within a target gene. Therefore, when fetuses within the uteri of pregnant murine females are exposed to these reagents, fetal cells incorporating them should show mutations in the target gene. To examine a possible genome editing of fetal cells in vivo, we intravenously administered a solution containing plasmid DNA-FuGENE complex to pregnant wild-type female mice [which had been successfully mated with enhanced green fluorescent protein (EGFP)-expressing male transgenic mice] on day 12.5 of gestation. The plasmid DNA induces the expression of gRNA, which was targeted at the EGFP cDNA, and that of the Cas9 gene. All fetuses in the pregnant females should express EGFP systemically, since they are heterozygous (Tg/+) for the transgene. Thus, the delivery of CRISPR system targeted at EGFP in the fetuses will cause a reduced expression of EGFP as a result of the genome editing of EGFP genomic sequence. Of the 24 fetuses isolated from three pregnant females 2 days after gene delivery, 3 were found to have reduced fluorescence in their hearts. Genotyping of the dissected hearts revealed the presence of the transgene construct (Cas9 gene) in all the samples. Furthermore, all the three samples exhibited mutations at the target loci, although normal cells were also present. Thus, transplacental delivery of gene editing components may be a useful tool for developing animal models with heart disorder for heart-related disease research, and gene therapy in congenital heart defects such as hypertrophic cardiomyopathy (HCM). © 2019 IUBMB Life, 9999(9999):1-10, 2019.
基因组编辑，以 CRISPR/Cas9 为例，现在被认为是一种强大的内源靶基因工程工具。它只使用两种成分，即 DNA、mRNA 或蛋白质形式的 Cas9 和针对靶基因的向导 RNA（gRNA）。当这些成分被转移到细胞中时，它们会在靶基因内产生插入/缺失突变（indels）。因此，当怀孕的雌性小鼠子宫内的胎儿暴露于这些试剂时，吸收它们的胎儿细胞应该在靶基因中出现突变。为了检查体内胎儿细胞的可能基因组编辑，我们在妊娠第 12.5 天向成功交配过表达增强型绿色荧光蛋白（EGFP）的转基因雄性小鼠的野生型雌性小鼠静脉内给予含有质粒 DNA-FuGENE 复合物的溶液[。质粒 DNA 诱导靶向 EGFP cDNA 和 Cas9 基因的 gRNA 的表达。由于转基因杂合子（Tg/+），所有怀孕雌性中的胎儿都应该全身表达 EGFP。因此，针对 EGFP 胎儿的 CRISPR 系统的递送将导致 EGFP 基因组序列的基因组编辑导致 EGFP 的表达减少。在基因递送后 2 天从 3 只怀孕雌性中分离的 24 只胎儿中，有 3 只心脏荧光减弱。对分离的心脏进行基因分型显示所有样本均存在转基因构建体（Cas9 基因）。此外，所有三个样本均在靶位点出现突变，尽管也存在正常细胞。因此，基因编辑成分的胎盘递送可能是开发用于心脏相关疾病研究的心脏疾病动物模型和肥厚型心肌病（HCM）等先天性心脏缺陷的基因治疗的有用工具。2019 年国际生物化学与分子生物学联合会生命杂志，9999（9999）：1-10，2019。
