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：
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
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/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

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.

项目摘要尽管基因组工程方面取得了非同寻常的进步，但所有人都需要精确有效的基因校正工具细胞类型和所需的编辑仍然缺乏。当前可编程的DNA裂解工具，例如CRISPR-CAS9，依靠细胞DNA修复机制，这些修复机制效率低下，在有丝分裂后细胞中不起作用。因此，基因组编辑仍然需要有效，可靠的工具，这些工具可以进行多种特定的DNA序列改变。这些工具可能在两个基本的生物学发现中都具有广泛的应用，从而允许新的方式筛查和治疗剂，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，生化表征和酶工程的发展基于积分酶的工具，用于多种细胞类型中大基因的可编程，多路复用插入独立于DNA修复。这些新整合酶蛋白的发现，表征和工程两者都将建立在我们的CRISPR酶发现的深刻历史上，并从新的高通量中汲取地雷生物多样性的方法。与发现这些新酶的发现互补，我们将结合基于CAS9的基因组编辑，具有整合酶工程，以开发可编程的多重基因组不依赖DNA修复机制的集成系统，允许将大序列整合在任何单元格类型。我们将探索输送机制，包括病毒，电穿孔和新型脂质纳米颗粒制剂以编辑T细胞和神经元。我们将设计集成酶的方面，包括蛋白质工程和现场诱变，以增加系统的活性并筛选许多插入位点为技术制定设计规则。此外，通过研究正交积分站点，我们可以开发插入工具的多路复用版本，可在给定单元格中编辑多达三个站点，其效率高于其他单位工具。我们将应用这些多路复用集成酶来开发一个新的筛选系统，在其中标记多个基因可用于确定高通量中的蛋白质相互作用伴侣。我们的新集成酶系统还将应用于开发多个编辑的T细胞，以改善免疫肿瘤疗法。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，并成为更广泛的宝贵资源生物医学研究。可编程基因集成与CRISPR恢复的集成酶将允许更多高级基因组工程应用将在细胞和体内进行，加速了生物医学研究，能够对基本的生物学过程和疾病机制进行更大的探索，以及促进新颖的治疗发展。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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
影响因子：
0
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.

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.

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.