Physiological role and mechanisms of action for a long noncoding haplotype region [RNA-Seq]

Direct targeting of noncoding genomic regions harboring common sequence variants associated with human traits through in vivo animal model studies and precise genome editing in human cells is essential for closing the critical gap between genetic discoveries and physiological understanding. However, such investigation has been impractical for most of these variants as they have small effect sizes and are in haplotypes containing multiple single nucleotide polymorphisms (SNPs) spanning thousands of base pairs. We developed an integrated approach to address this challenge, combining an efficient two-step technique to precisely edit large haplotypes in human induced pluripotent stem cells and orthologous region deletion in phenotypically permissive animal models. As proof of principle, we applied this approach to examine a blood pressure associated locus with a noncoding haplotype containing 11 SNPs spanning 17.4 kbp. We found a robust blood pressure effect of nearly 10 mmHg and identified the physiological and molecular mechanisms involved.通过体内动物模型研究与人类细胞精准基因组编辑，直接靶向携带与人类性状相关常见序列变异的非编码基因组区域，对于填补遗传发现与生理认知之间的关键空白至关重要。然而，此类变异多数效应量较小，且处于跨度达数千碱基对、包含多个单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）的单体型区域中，因此针对绝大多数此类变异开展此类研究并不现实。为此我们开发了一套整合研究方案以应对该挑战，将两项高效技术相结合：一是在人类诱导多能干细胞（human induced pluripotent stem cells）中精准编辑大型单体型的两步法技术，二是在表型兼容的动物模型中进行同源区域缺失操作的技术。作为原理验证，我们将该方案应用于一个血压相关基因座的研究，该基因座的非编码单体型包含11个单核苷酸多态性，跨度达17.4千碱基对（kilobase pair, kbp）。我们观测到了近10毫米汞柱（millimeter of mercury, mmHg）的显著血压效应，并阐明了其所涉及的生理与分子机制。