Programmable gene integration and cell engineering with CRISPR-directed integrases

使用 CRISPR 引导的整合酶进行可编程基因整合和细胞工程

基本信息

批准号：
10491366
负责人：
Omar O Abudayyeh
金额：
$ 57.4万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-20 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10491366
关键词：
Address Bacteriophages Biochemical Biodiversity Bioinformatics Biological Biological Assay Biological Process Biology Biomedical Research CRISPR/Cas technology Cell Line Cell Therapy Cell model Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex Confocal Microscopy DNA DNA Repair DNA Sequence Alteration Development Disease Disease model Electroporation Elements Engineering Enzymes Formulation Future Genes Genetic Variation Genome Genome engineering Guide RNA Immune Immunooncology Integrase Interphase Cell Location Mammalian Cell Mediating Methodology Mining Mitotic Modality Modification Mutagenesis Mutation Neurons Pathway interactions Positioning Attribute Protein Engineering Proteins RNA Recording of previous events Regulatory Element Resources Site Study models System Systems Integration T-Lymphocyte Technology Testing Therapeutic Therapeutic Uses Virus Work base biological research cell type cellular engineering computational pipelines cytokine design epigenome gene correction genome editing human disease improved in vitro Model in vivo innovation insertion/deletion mutation insight lipid nanoparticle new technology novel novel therapeutics prime editing programs protein protein interaction recruit repaired response screening stress granule therapeutic development tool transcription factor

项目摘要

Project Summary Despite extraordinary advances in genome engineering, tools for precise and efficient gene correction across all cell types and desired edits remain lacking. Current programmable DNA cleavage tools, such as CRISPR-Cas9, rely on cellular DNA repair mechanisms, which are inefficient and do not function in post-mitotic cells. Thus, genome editing still needs efficient, robust tools that can make a variety of specific DNA sequence alterations. These tools could have broad applications across both basic biological discovery, allowing for new modalities of screening, and therapeutics, including engineered cell therapies. The proposed work will address these needs by combining computational discovery, biochemical characterization, and enzyme engineering to develop integrase-based tools for programmable, multiplexed insertion of large genes in diverse cell types independent of DNA repair. The discovery, characterization, and engineering of these new integrase proteins will both build upon our deep history of CRISPR enzyme discovery, as well as draw from new, high-throughput approaches to mine biological diversity. Complementary to the discovery of these new enzymes, we will combine Cas9-based genome editing with integrase engineering to develop programmable, multiplexed genome integration systems that do not depend on DNA repair mechanisms, allowing integration of large sequences in any cell type. We will explore delivery mechanisms, including viruses, electroporation, and novel lipid nanoparticle formulations to edit T cells and neurons. We will engineer aspects of the integrases, including protein engineering and site mutagenesis, to boost activity of the system and screen many insertion sites to develop design rules for the technology. Moreover, through studying orthogonal integrases sites we can develop multiplexed versions of the insertion tool to edit up to three sites in a given cell with superior efficiency over other tools. We will apply these multiplexed integrases to develop a new screening system, where tagging of multiple genes can be used for determining protein interaction partners in high throughput. Our new integrase systems will also be applied to the development of multiple-edited T-cells for improved immuno-oncology therapies. The multiple technologies resulting from these discoveries and engineering efforts will overcome the limitations of existing genome and epigenome engineering approaches and serve as a valuable resource for broader biomedical research. Programmable gene integration with CRISPR-recruited integrases will allow for more advanced genome engineering applications to be pursued in cells and in vivo, accelerating the pace of biomedical research, enabling greater exploration of basic biological processes and disease mechanisms, and promoting novel therapeutic developments.

项目概要尽管基因组工程取得了巨大进步，但用于精确、高效基因校正的工具仍然存在。细胞类型和所需的编辑仍然缺乏。当前的可编程DNA切割工具，例如CRISPR-Cas9，依赖细胞 DNA 修复机制，但这种机制效率低下，并且在有丝分裂后细胞中不起作用。因此，基因组编辑仍然需要高效、强大的工具来进行各种特定的 DNA 序列改变。这些工具可以在基本生物发现方面具有广泛的应用，从而允许新的模式筛选和治疗，包括工程细胞疗法。拟议的工作将满足这些需求通过结合计算发现、生化表征和酶工程来开发基于整合酶的工具，用于在不同细胞类型中可编程、多重插入大基因独立于 DNA 修复。这些新整合酶蛋白的发现、表征和工程设计都将建立在我们 CRISPR 酶发现的深厚历史之上，并借鉴新的高通量挖掘生物多样性的方法。作为对这些新酶的发现的补充，我们将结合基于 Cas9 的基因组编辑与整合酶工程，以开发可编程、多重基因组不依赖于 DNA 修复机制的整合系统，允许将大序列整合到任何细胞类型。我们将探索递送机制，包括病毒、电穿孔和新型脂质用于编辑 T 细胞和神经元的纳米颗粒制剂。我们将设计集成的各个方面，包括蛋白质工程和位点诱变，以增强系统的活性并筛选许多插入位点制定技术的设计规则。此外，通过研究正交整合酶位点，我们可以开发插入工具的多路复用版本可在给定单元中编辑最多三个位点，其效率优于其他工具工具。我们将应用这些多重整合酶来开发一种新的筛选系统，其中标记多个基因可用于高通量确定蛋白质相互作用伙伴。我们的新整合酶系统还将应用于开发多重编辑的 T 细胞，以改进免疫肿瘤疗法。这这些发现和工程努力产生的多种技术将克服现有的基因组和表观基因组工程方法，可以作为更广泛的有价值的资源生物医学研究。与 CRISPR 招募的整合酶进行可编程基因整合将允许更多在细胞和体内追求先进的基因组工程应用，加快了生物医学研究，能够更好地探索基本生物过程和疾病机制，以及促进新的治疗方法的发展。