Core C: Genome Engineering Core

核心 C：基因组工程核心

• 批准号: 10006187
• 负责人: Bruce R Conklin
• 金额: $ 28.35万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006187
• 关键词: Adopted; Affect; Antibiotic Resistance; Antibiotics; Biological Process; CRISPR interference; CRISPR/Cas technology; Cardiac; Cardiac Myocytes; Cardiac development; Cell Line; Cells; Chromatin; Chromatin Remodeling Factor; Cicatrix; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Damage; DNA Methylation; Disease; Distant; Emerging Technologies; Engineering; Enhancers; Epigenetic Process; Event; GATA4 gene; Gene Deletion; Gene Expression; Gene Expression Regulation; Gene Silencing; Generations; Genes; Genetic; Genome; Genome engineering; Growth; Heart Diseases; Human; Individual; Laboratories; Location; Mediating; Methods; Methylation; Modification; Molecular; Mutation; Mutation Detection; Nonhomologous DNA End Joining; Nuclear Pore Complex; Open Reading Frames; Point Mutation; Protein Complex Subunit; Proteins; RNA Splicing; Reagent; Regulation; Research Personnel; Research Project Grants; Role; Services; Single base substitution; Site; Techniques; Technology; Testing; Transgenes; Update; Validation; Variant; Yin; base; cardiogenesis; chromatin modification; combinatorial; congenital heart disorder; design; digital; epigenome editing; experience; genome editing; improved; induced pluripotent stem cell; insertion/deletion mutation; interest; knockout gene; mutant; new technology; novel strategies; nuclease; protein complex; protein function; repaired; tool; transcription activator-like effector nucleases

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.

项目摘要/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。我们将开发并采用CRISPR/CAS9介导的基因删除的新兴技术， 单基础变化，转化插入和表观遗传重塑。 PPG提案利用了高级 基因组工程技术。我们将专门提供人类IPSC的基因组工程服务 有效地交付了用于心脏分化的工程IPSC。基因组工程核心也将 提供表观基因组编辑方法，可修饰细胞的基因组而无需切割DNA。 crispr- 相关的核酸酶（CAS9）已修饰，因此核酸酶在制造“ DCA”现在不活跃的情况下可以 被用来携带和将各种生物活性分子定位到基因组中的任何位置。我们 开创了使用CRISPRI的使用，即在IPSC中沉默基因表达，并参与更好的发展 激活基因表达的方法（CRISPRA）。 我们的团队是基因组工程领域的知名领导者，并努力改善 人IPSC中的基因组编辑，以使PPG研究人员揭示分子基础的努力。 先天性心脏病。在过去的五年中，我们制作了> 50种不同的基因修饰的人IPSC线 通过精确模仿疾病突变的点突变，产生了基因的插入/缺失（indels） 敲除或引入内源基因标签以用于蛋白质功能的分子研究（Miyaoka等，2014， Huebsch等，2015； Mandegar等，2016；法官等，2017）。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/删除（indels）或删除基因的离散部分，以使其在IPSC中失活 就像在IPSC衍生的心肌细胞中一样，由于RNP介导的基因组编辑在我们的中更有效和准确 经验。此外，基因组工程核心将为 插入内源基因座的转基因，以及为PPG开发新的CRISPR方法 研究人员进一步研究心脏相互作用组。基因组工程核心将发展 通过不断更新的技术向PPG项目运送高质量工程IPSC的管道 回答心脏发展中的重要问题。