Transplacental Delivery of Genome Editing Components Causes Mutations in Embryonic Cardiomyocytes of Mid-Gestational Murine Fetuses
Transplacental Delivery of Genome Editing Components Causes Mutations in Embryonic Cardiomyocytes of Mid-Gestational Murine Fetuses
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基因组编辑成分的经胎盘递送导致妊娠中期鼠胎儿胚胎心肌细胞突变
DOI：
10.1002/iub.2004
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发表时间：
2019
期刊：
影响因子：
4.6
通讯作者：
Sato Masahiro
中科院分区：
文献类型：
--
作者：
Nakamura Shingo;Ishihara Masayuki;Ando Naoko;Watanabe Satoshi;Sakurai Takayuki;Sato Masahiro
以CRISPR/Cas9为例的基因组编辑现在被认为是内源性靶基因工程的有力工具。它只使用两种组分，即DNA、mRNA或蛋白质形式的Cas9;和对靶基因特异性的指导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，当妊娠雌性小鼠子宫内的胎儿暴露于这些试剂时，掺入这些试剂的胎儿细胞应显示靶基因突变。为了检查体内胎儿细胞可能的基因组编辑，我们在妊娠第12.5天向妊娠野生型雌性小鼠[已成功与表达增强型绿色荧光蛋白（EGFP）的雄性转基因小鼠交配]静脉内给予含有质粒DNA-FuGENE复合物的溶液。质粒DNA诱导靶向EGFPcDNA的gRNA和Cas9基因的表达。妊娠雌性中的所有胎儿都应全身表达EGFP，因为它们对于转基因是杂合的（Tg/+）。因此，在胎儿中递送靶向EGFP的CRISPR系统将由于EGFP基因组序列的基因组编辑而导致EGFP表达减少。在基因递送后2天从三个怀孕雌性分离的24个胎儿中，发现3个在其心脏中具有减少的荧光。解剖心脏的基因分型揭示了所有样品中转基因构建体（Cas9基因）的存在。此外，尽管也存在正常细胞，但所有三个样品都在靶基因座处表现出突变。因此，基因编辑组件的经胎盘递送可能是开发心脏疾病动物模型的有用工具，用于心脏相关疾病研究，以及先天性心脏缺陷如肥厚性心肌病（HCM）的基因治疗。© 2019 IUBMB Life，9999（9999）：1-10，2019
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 theEGFPcDNA, and that of theCas9gene. 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 atEGFPin the fetuses will cause a reduced expression of EGFP as a result of the genome editing ofEGFPgenomic 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 (Cas9gene) 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