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

• 批准号: 10672995
• 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
• 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/10672995
• 关键词: Acceleration; 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; Modality; Modification; Mutagenesis; Mutation; Neurons; Pathway interactions; Positioning Attribute; Protein Engineering; Proteins; RNA; Recording of previous events; Regulatory Element; Resources; Site; System; Systems Integration; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic Uses; Virus; Visualization; Work; biological research; cell type; cellular engineering; computational pipelines; cytokine; design; epigenome; gene correction; genome editing; human disease; improved; in vitro Model; in vivo; innovation; insight; lipid nanoparticle; new technology; novel; novel therapeutics; postmitotic; prime editing; protein protein interaction; recruit; repaired; response; screening; stress granule; therapeutic development; tool; transcription factor

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.

项目摘要 尽管基因组工程取得了非凡的进步，但在所有领域进行精确和有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。目前的可编程DNA切割工具，如CRISPR-Cas9， 依赖于细胞DNA修复机制，这是低效的，在有丝分裂后的细胞中不起作用。因此，在本发明中， 基因组编辑仍然需要高效，强大的工具，可以进行各种特定的DNA序列改变。 这些工具可以广泛应用于基础生物学发现，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，生物化学表征和酶工程来开发 用于在不同细胞类型中可编程、多重插入大基因的基于整合酶的工具 独立于DNA修复。这些新整合酶蛋白的发现、表征和工程化 将建立在我们CRISPR酶发现的深厚历史基础上， 对生物多样性的保护。作为发现这些新酶的补充，我们将联合收割机 基于Cas9的基因组编辑与整合酶工程，以开发可编程的多重基因组 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。我们将探索传递机制，包括病毒，电穿孔和新型脂质体。 纳米颗粒制剂来编辑T细胞和神经元。我们将设计集成的各个方面，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， 为这项技术制定设计规则。此外，通过研究正交积分酶位点， 插入工具的多路复用版本以在给定细胞中编辑多达三个位点，其具有比其它插入工具更上级的效率。 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， 基因可用于以高通量确定蛋白质相互作用配偶体。我们的新型整合酶系统 还将应用于开发多重编辑的T细胞，以改善免疫肿瘤学疗法。的 从这些发现和工程努力中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，并作为更广泛的宝贵资源， 生物医学研究使用CRISPR募集的整合酶进行可编程基因整合将允许更多的 先进的基因组工程应用将在细胞和体内进行，加快步伐， 生物医学研究，使更多的探索基本生物过程和疾病机制， 促进新的治疗发展。

项目成果

Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.

可编程RNA引导的核酸内切酶广泛存在于真核生物及其病毒中。

• DOI: 10.1101/2023.06.13.544871
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Jiang,Kaiyi;Lim,Justin;Sgrizzi,Samantha;Trinh,Michael;Kayabolen,Alisan;Yutin,Natalya;Koonin,EugeneV;Abudayyeh,OmarO;Gootenberg,JonathanS
• 通讯作者: Gootenberg,JonathanS

Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases.

• DOI: 10.1038/s41587-022-01527-4
• 发表时间: 2023-04
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Yarnall, Matthew T. N.;Ioannidi, Eleonora I.;Schmitt-Ulms, Cian;Krajeski, Rohan N.;Lim, Justin;Villiger, Lukas;Zhou, Wenyuan;Jiang, Kaiyi;Garushyants, Sofya K.;Roberts, Nathaniel;Zhang, Liyang;Vakulskas, Christopher A.;Walker, John A. I. I. I. I.;Kadina, Anastasia P.;Zepeda, Adrianna E.;Holden, Kevin;Ma, Hong;Xie, Jun;Gao, Guangping;Foquet, Lander;Bial, Greg;Donnelly, Sara K.;Miyata, Yoshinari;Radiloff, Daniel R.;Henderson, Jordana M.;Ujita, Andrew;Abudayyeh, Omar O.;Gootenberg, Jonathan S.
• 通讯作者: Gootenberg, Jonathan S.

Programmable RNA-guided DNA endonucleases are widespread in eukaryotes and their viruses.

• DOI: 10.1126/sciadv.adk0171
• 发表时间: 2023-09-29
• 期刊: SCIENCE ADVANCES
• 影响因子: 13.6
• 作者: Jiang, Kaiyi;Lim, Justin;Sgrizzi, Samantha;Trinh, Michael;Kayabolen, Alisan;Yutin, Natalya;Bao, Weidong;Kato, Kazuki;Koonin, Eugene V.;Gootenberg, Jonathan S.;Abudayyeh, Omar O.
• 通讯作者: Abudayyeh, Omar O.

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox.

• DOI: 10.1021/acs.biochem.3c00159
• 发表时间: 2023-12-19
• 期刊: BIOCHEMISTRY
• 影响因子: 2.9
• 作者: Koonin, Eugene V.;Gootenberg, Jonathan S.;Abudayyeh, Omar O.
• 通讯作者: Abudayyeh, Omar O.