Centre for Organismal Studies, Heidelberg University, 69120 Heidelberg, Germany.
Department of Pediatric Cardiology, University Hospital Heidelberg, 69120 Heidelberg, Germany.
Dis Model Mech. 2023 Aug 1;16(8). doi: 10.1242/dmm.049811. Epub 2023 Aug 16.
High-throughput DNA sequencing studies increasingly associate DNA variants with congenital heart disease (CHD). However, functional modeling is a crucial prerequisite for translating genomic data into clinical care. We used CRISPR-Cas9-mediated targeting of 12 candidate genes in the vertebrate model medaka (Oryzias latipes), five of which displayed a novel cardiovascular phenotype spectrum in F0 (crispants): mapre2, smg7, cdc42bpab, ankrd11 and myrf, encoding a transcription factor recently linked to cardiac-urogenital syndrome. Our myrf mutant line showed particularly prominent embryonic cardiac defects recapitulating phenotypes of pediatric patients, including hypoplastic ventricle. Mimicking human mutations, we edited three sites to generate specific myrf single-nucleotide variants via cytosine and adenine base editors. The Glu749Lys missense mutation in the conserved intramolecular chaperon autocleavage domain fully recapitulated the characteristic myrf mutant phenotype with high penetrance, underlining the crucial function of this protein domain. The efficiency and scalability of base editing to model specific point mutations accelerate gene validation studies and the generation of human-relevant disease models.
高通量 DNA 测序研究越来越多地将 DNA 变体与先天性心脏病 (CHD) 联系起来。然而，功能建模是将基因组数据转化为临床护理的关键前提。我们使用 CRISPR-Cas9 介导的方法在脊椎动物模型斑马鱼 (Oryzias latipes) 中靶向 12 个候选基因，其中 5 个在 F0 (crispants) 中显示出一种新颖的心血管表型谱：mapre2、smg7、cdc42bpab、ankrd11 和 myrf，编码最近与心脏-泌尿生殖综合征相关的转录因子。我们的 myrf 突变体系表现出特别明显的胚胎心脏缺陷，再现了儿科患者的表型，包括心室发育不良。通过胞嘧啶和腺嘌呤碱基编辑器，我们模拟人类突变，编辑了三个位点以产生特定的 myrf 单核苷酸变体。保守的分子内伴侣自切割结构域中的 Glu749Lys 错义突变完全再现了具有高穿透性的特征性 myrf 突变体表型，突出了该蛋白结构域的关键功能。碱基编辑模拟特定点突变的效率和可扩展性加速了基因验证研究和人类相关疾病模型的产生。
