Core C: Genome Engineering Core

项目概要/摘要 核心C -基因组工程核心 Core C专注于为心脏研究提供最新的基因组工程技术， 发展我们将开发和采用CRISPR/Cas9介导的基因缺失新兴技术， 单碱基改变、转基因插入和表观遗传重塑。PPG提案采用先进的 基因工程技术。我们将专门提供人类iPSCs的基因组工程服务， 高效地递送工程化的iPSC用于心脏分化。基因组工程核心还将 提供表观基因组编辑方法，在不切割DNA的情况下修改细胞的基因组。CRISPR- 相关核酸酶（Cas9）已经被修饰，使得核酸酶在制造“dCas”中是无活性的， 用于携带和定位多种生物活性分子到基因组中的任何位置。我们 率先使用CRISPRi沉默iPSCs中的基因表达，并参与开发更好的 激活基因表达的方法（CRISPRa）。 我们的团队是基因组工程领域公认的领导者，并努力改善基因组工程的各个方面。 人类iPSC中的基因组编辑，以使PPG研究人员能够努力解开分子基础 先天性心脏病在过去的5年里，我们制造了超过50种不同的转基因人类iPSC系， 用精确模拟疾病突变的点突变，产生基因的插入/缺失（indels）， 敲除或引入内源基因标签用于蛋白质功能的分子研究（Miyaoka等人，2014, Huebsch等人，2015年，Mandegar等人，2016，Judge等人，2017年）。基因组工程核心将采用 基因组工程的最新方法，例如使用CRISPR/Cas9 RNP-蛋白质复合物（RNP） 引入插入/缺失（indels），或删除基因的离散部分，以使其在iPSC中也能表达 与iPSC衍生的心肌细胞一样，由于RNP介导的基因组编辑在我们的研究中更有效和准确， 体验.此外，基因组工程核心将提供基因组工程服务， 在内源基因座插入转基因，以及开发用于PPG的新CRISPR方法 研究人员进一步研究心脏相互作用组。基因组工程核心将开发一个 通过不断更新的技术，为PPG项目提供高质量的工程iPSC， 回答心脏发育中的重要问题。

PROJECT SUMMARY/ABSTRACT CORE C – GENOME ENGINEERING CORE Core C is focused on providing the most up-to-date genome engineering technology for studies of cardiac development. We will develop and adopt emerging technology for CRISPR/Cas9-mediated gene deletions, single-base changes, transgene insertions, and epigenetic remodeling. The PPG proposal utilizes advanced genome engineering techniques. We will specifically provide genome engineering services of human iPSCs to efficiently deliver engineered iPSCs for cardiac differentiation. The Genome Engineering Core will also provide epigenome editing methods, that modify the cell's genome without cutting DNA. The CRISPR- associated nuclease (Cas9) has been modified so that the nuclease is inactive in making “dCas” that can now be used to carry and localize a wide variety of bioactive molecules to any location in the genome. We pioneered the use of CRISPRi that silences gene expression in iPSCs, and are involved in developing better methods that activate gene expression (CRISPRa). Our team is an established leader in genome engineering, and has made efforts to improve every aspect of genome editing in human iPSCs to benefit the PPG investigators in their efforts to unravel the molecular basis of congenital heart disease. In the last 5 years, we made >50 different genetically modified human iPSC lines with point mutations that exactly mimic the disease mutations, generate insertions/deletions (indels) for gene knockouts, or introduce endogenous gene tags for molecular studies of protein function (Miyaoka et al., 2014, Huebsch et al., 2015, Mandegar et al., 2016, Judge et al., 2017). The Genome Engineering Core will adopt the latest methods for genome engineering, such as the use of CRISPR/Cas9 RNP-protein complexes (RNP) to introduce insertion/deletions (indels), or delete discrete portions of genes to inactivate them in iPSCs as well as in iPSC-derived cardiomyocytes, since RNP-mediated genome editing is more efficient and accurate in our experience. In addition, the Genome Engineering Core will provide genome engineering services for the insertion of transgenes at endogenous loci, as well as develop new CRISPR methods for the PPG investigators to further investigate the cardiac interactome. The Genome Engineering Core will develop a pipeline to deliver high-quality engineered iPSCs to the PPG projects with continuously updated techniques, to answer vital questions in heart development.