Engineering Efficient and Controllable Base Editors

工程高效且可控的碱基编辑器

基本信息

批准号：
10796080
负责人：
Rahul Manu Kohli
金额：
$ 12.16万
依托单位：
UNIVERSITY OF PENNSYLVANIA
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：
2021-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10796080
关键词：
Address Adenosine Antibodies Antibody Response Attenuated B-Lymphocytes BCAR1 gene Basic Science Biochemical Biological Biological Models Biology Biotechnology Chromosomal translocation Clustered Regularly Interspaced Short Palindromic Repeats Complex Coupled Cytosine Cytosine deaminase DNA Deaminase Deamination Diagnosis Disease Distant Endonuclease I Engineering Enzymes Epigenetic Process Event Family Family member Generations Genes Genetic Engineering Genome Genome Stability Genome engineering Genomic Instability Genomics Guide RNA Hyperactivity Immune Immunoglobulin Somatic Hypermutation Immunoglobulins Impairment Knock-out Knowledge Laboratories Link Malignant Neoplasms Medicine Molecular Mutagenesis Mutate Mutation Nature Nucleic Acids Nucleosides Oncogenic Outcome Pathway interactions Patients Physiological Play Positioning Attribute Property RNA RNA Binding Rattus Reaction Resistance Retroviridae Risk Role Single-Stranded DNA Site Structure System T-Lymphocyte Terminator Codon Therapeutic Time Transfer RNA Translating Translational Research Uracil Variant Work Zinc activation-induced cytidine deaminase anti-cancer anticancer activity apoB mRNA editing catalytic subunit base base editing base editor chemotherapy chimeric antigen receptor chimeric antigen receptor T cells cost disease-causing mutation ds-DNA frontier genome editing genomic locus improved innovation insertion/deletion mutation insight member next generation precise genome editing programs reconstitution repaired risk minimization small molecule spatiotemporal tool transition mutation

项目摘要

PROJECT SUMMARY This proposal aims to manipulate DNA deaminase enzymes to generate hyperactive and controllable base editors that can be targeted for precise gene editing. Base editing of the immunoglobulin locus by AID, the ancestral member of the AID/APOBEC family of cytosine deaminase enzymes, normally initiates maturation of antibody responses in B-cells, while APOBECs provide protection against retroviruses. Out of their physiological context, when DNA deaminases are directed by catalytically-impaired CRIPSR/Cas proteins, their base editing activity can be used to introduce targeted mutations at a desired genomic locus. While this system offers a potentially powerful means to edit the genome for biological or therapeutic purposes, base editors have two barriers that limit their broader application in basic and translational research. First, DNA deaminases have naturally evolved to be constrained enzymes with low overall catalytic activity, as hyperactivation is associated with increased oncogenic mutations. Second, when dysregulated, AID/APOBECs are known to act outside of their targets, promoting cancer mutagenesis, chromosomal translocations, and resistance to chemotherapy. Given that natural regulatory constraints on DNA deaminases are lost in base editor complexes, these constructs pose similar risks to the genome. In this proposal, we harness our extensive knowledge of the mechanism, structure and function of deaminase enzymes in order to overcome these challenges. For one, hyperactive deaminases have been generated to overcome the naturally attenuated activity, and we will exploit these variants to evaluate the hypothesis that increasing the deamination rate can improve the efficiency of the base editing reaction, while simultaneously improving precision. Second, we have devised split deaminases that can only be reconstituted at the targeted locus under the control of a small molecule. This strategy newly offers spatiotemporal control, a critical requirement that will facilitate the use of base editors in the lab and is essential to therapeutic applications in patients. Given the wide range of potential uses for base editors, we will demonstrate the importance of efficiency and control broadly across diverse genomic sites, and then specifically by generating enhanced chimeric antigen receptor expressing T (CAR-T) cells as a model system. The tools developed here will globally advance deaminases as base editors and will readily translate to other innovations in CRISPR/Cas proteins, and to genome engineering more generally.

项目概要该提案旨在操纵DNA脱氨酶以产生高度活跃且可控的碱基可以有针对性地进行精确基因编辑的编辑器。 AID 对免疫球蛋白位点进行碱基编辑胞嘧啶脱氨酶 AID/APOBEC 家族的祖先成员，通常启动 B 细胞中的抗体反应，而 APOBEC 则提供针对逆转录病毒的保护。出于他们的生理上下文中，当 DNA 脱氨酶受到催化受损的 CRIPSR/Cas 蛋白指导时，它们的碱基编辑活性可用于在所需的基因组位点引入靶向突变。虽然该系统提供了碱基编辑器是出于生物学或治疗目的编辑基因组的潜在强大手段，碱基编辑器有两种限制其在基础研究和转化研究中更广泛应用的障碍。首先，DNA脱氨酶自然进化为具有低总体催化活性的受限酶，因为过度激活是相关的随着致癌突变的增加。其次，当失调时，AID/APOBEC 会在外部发挥作用它们的目标是促进癌症突变、染色体易位和化疗耐药性。鉴于 DNA 脱氨酶的自然调控限制在碱基编辑器复合物中消失，这些构建体对基因组造成类似的风险。在这个提案中，我们利用我们对该机制的广泛了解，脱氨酶的结构和功能，以克服这些挑战。其一，过度活跃脱氨酶的产生是为了克服自然减弱的活性，我们将利用这些变体评估增加脱氨基速率可以提高碱基编辑效率的假设反应，同时提高精度。其次，我们设计了分裂脱氨酶，只能在小分子的控制下在目标位点重建。该策略新提供时空控制，一项关键要求，将有助于在实验室中使用碱基编辑器，并且至关重要到患者的治疗应用。鉴于碱基编辑器的广泛潜在用途，我们将证明跨不同基因组位点的效率和控制的重要性，然后具体通过生成增强型嵌合抗原受体表达 T (CAR-T) 细胞作为模型系统。工具这里开发的技术将在全球范围内推进脱氨酶作为碱基编辑的发展，并很容易转化为其他创新 CRISPR/Cas 蛋白，以及更广泛的基因组工程。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

CRISPR tiling screen reveals cancer epigenetic 'Goldilocks' state.

CRISPR 平铺筛选揭示了癌症表观遗传“金发姑娘”状态。

DOI：
发表时间：
2023-09
期刊：
影响因子：
13.8
作者：
Barka, Aleksia;Kohli, Rahul M;Shi, Junwei
通讯作者：
Shi, Junwei

Engineering enhanced CAR T-cells for improved cancer therapy.

工程增强 CAR T 细胞以改善癌症治疗。

DOI：
发表时间：
2021-08
期刊：
影响因子：
22.7
作者：
Milone, Michael C;Xu, Jie;Chen, Sai;Collins, McKensie A;Zhou, Jianfeng;Powell Jr, Daniel J;Melenhorst, J Joseph
通讯作者：
Melenhorst, J Joseph

The Base-Editing Enzyme APOBEC3A Catalyzes Cytosine Deamination in RNA with Low Proficiency and High Selectivity.

碱基编辑酶 APOBEC3A 以低效率和高选择性催化 RNA 中的胞嘧啶脱氨基。

DOI：
发表时间：
2022-03-18
期刊：
影响因子：
4
作者：
Barka, Aleksia;Berríos, Kiara N;Bailer, Peter;Schutsky, Emily K;Wang, Tong;Kohli, Rahul M
通讯作者：
Kohli, Rahul M

Controllable genome editing with split-engineered base editors.

使用拆分工程碱基编辑器进行可控基因组编辑。

DOI：
发表时间：
2021-12
期刊：
影响因子：
14.8
作者：
Berríos, Kiara N;Evitt, Niklaus H;DeWeerd, Rachel A;Ren, Diqiu;Luo, Meiqi;Barka, Aleksia;Wang, Tong;Bartman, Caroline R;Lan, Yemin;Green, Abby M;Shi, Junwei;Kohli, Rahul M
通讯作者：
Kohli, Rahul M

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors.

Cas9 和高度活跃的 AID 之间的协作在碱基编辑器中建立了广泛且多样化的突变足迹。

DOI：
发表时间：
2024-02-28
期刊：
影响因子：
14.9
作者：
Berríos, Kiara N;Barka, Aleksia;Gill, Jasleen;Serrano, Juan C;Bailer, Peter F;Parker, Jared B;Evitt, Niklaus H;Gajula, Kiran S;Shi, Junwei;Kohli, Rahul M
通讯作者：
Kohli, Rahul M