Core C: Genome Engineering Core

核心 C：基因组工程核心

• 批准号: 10471986
• 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/10471986
• 关键词: Adopted; Affect; Antibiotic Resistance; Antibiotics; Biological Process; CRISPR interference; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Cardiac; 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; gene network; genome editing; improved; induced pluripotent stem cell; induced pluripotent stem cell derived cardiomyocytes; 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 我们将开发和采用 CRISPR/Cas9 介导的基因删除新兴技术， PPG 提案利用了先进的单碱基改变、转基因插入和表观遗传重塑。 我们将专门提供人类 iPSC 的基因组工程服务。 基因组工程核心也将有效地提供用于心脏分化的工程 iPSC。 提供表观基因组编辑方法，无需切割 DNA 即可修改细胞基因组。 相关核酸酶 (Cas9) 已被修改，因此该核酸酶在制造“dCas”时不具有活性，现在可以 可用于将多种生物活性分子携带和定位到基因组中的任何位置。 率先使用 CRISPRi 沉默 iPSC 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 的基因组编辑有利于 PPG 研究人员解开分子基础的努力 在过去 5 年里，我们培育了超过 50 种不同的转基因人类 iPSC 系。 通过精确模拟疾病突变的点突变，生成基因插入/删除 (indels) 敲除，或引入内源基因标签以进行蛋白质功能的分子研究（Miyaoka 等，2014， Huebsch 等人，2015，Mandegar 等人，2016，Judge 等人，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 (indel)，或删除基因的离散部分以使其在 iPSC 中失活 与 iPSC 衍生的心肌细胞一样，因为 RNP 介导的基因组编辑在我们的研究中更加有效和准确 此外，基因组工程核心将为人类提供基因组工程服务。 在内源基因座插入转基因，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，基因组工程核心将开发一个 通过不断更新的技术向 PPG 项目提供高质量的工程 iPSC 的管道， 回答心脏发育中的重要问题。