Programmable gene integration and cell engineering with CRISPR-directed integrases

项目摘要 尽管基因组工程取得了非凡的进步，但用于精确和高效的基因校正的工具 仍然缺乏单元格类型和所需的编辑。目前的可编程DNA切割工具，如CRISPR-Cas9， 依赖于细胞DNA修复机制，这种机制效率低下，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序列更改。 这些工具可以在两个基本的生物发现中得到广泛的应用，允许新的模式 筛查和治疗，包括工程细胞疗法。拟议的工作将满足这些需求 通过将计算发现、生化表征和酶工程相结合来开发 基于整合酶的工具，用于在不同类型的细胞中可编程、多路插入大基因 不依赖于DNA修复。这些新整合酶蛋白的发现、鉴定和工程 都将建立在我们深厚的CRISPR酶发现历史基础上，以及从新的、高通量的 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的基因组编辑和整合酶工程，以开发可编程的多路基因组 不依赖DNA修复机制的整合系统，允许在 任何单元格类型。我们将探索递送机制，包括病毒、电穿孔和新型脂质 编辑T细胞和神经元的纳米颗粒配方。我们将设计整合酶的各个方面，包括 蛋白质工程和定点突变，以提高系统的活性，并筛选许多插入位点以 为这项技术制定设计规则。此外，通过研究正交整合酶位点，我们可以开发出 插入工具的多路复用版本，可在给定单元格中编辑最多三个站点，效率高于其他站点 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 还将用于开发多重编辑的T细胞，以改进免疫肿瘤学治疗。这个 这些发现和工程工作产生的多种技术将克服以下限制 现有的基因组和表观基因组工程方法，并作为更广泛的 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 先进的基因组工程应用将在细胞和体内进行，加快 生物医学研究，使人们能够更好地探索基本的生物过程和疾病机制；以及 促进新的治疗发展。

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.