Enhancing Genome Editing Technology with Natural Cas9 Inhibitors

利用天然 Cas9 抑制剂增强基因组编辑技术

• 批准号: 10092186
• 负责人: ERIK J. SONTHEIMER
• 金额: $ 35.06万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-13 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092186
• 关键词: Address; Automobile Driving; Bacteriophages; Basic Science; Binding; Bioinformatics; Biological; Biological Sciences; Biotechnology; CRISPR interference; CRISPR/Cas technology; Cell Culture Techniques; Cell Cycle; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Sequence Alteration; Development; Exhibits; Family; Genes; Genome engineering; Genomics; Goals; Guide RNA; Hereditary Disease; Homologous Gene; Human; Human Genome; Immune system; Immunity; Individual; Mammalian Cell; Measures; MicroRNAs; Mobile Genetic Elements; Modeling; Motivation; Neisseria meningitidis; Orthologous Gene; Performance; Phase; Phylogenetic Analysis; Phylogeny; Proteins; RNA; Reporting; Research; Risk; Safety; Science; Site; Specificity; System; Technology; Testing; Therapeutic; Time; Tissues; Work; base; cell type; clinical development; clinical practice; experimental study; flexibility; genome editing; improved; inhibitor/antagonist; knockout gene; mammalian genome; next generation; novel; nuclease; potency testing; pre-clinical; preclinical safety; prevent; programs; promoter; public health relevance; repaired; research and development; therapy development; tool

Modified Project Summary/Abstract Section CRISPR-Cas9 is revolutionizing the life sciences through its RNA-guided ability to target individual genes for disruption or for precise editing. In addition to accelerating basic research and biotechnology, Cas9 and its RNA guides also have the potential to transform the treatment of inherited diseases via genome editing-based cures, especially if delivery hurdles can be overcome and clinical safety can be proven. There are three subtypes of Cas9 systems (Types II-A, -B and -C), and all three have yielded Cas9 orthologs with demonstrated utility in mammalian genome editing. Our own work has established the Type II-C Cas9 from N. meningitidis (NmeCas9) as a compact, naturally hyperaccurate genome editing platform. Some of the safety concerns in Cas9’s clinical development arise from Cas9 activity that is excessive, prolonged, or present in unintended cell types. These concerns have been heightened by a shortage of effective tools that inactivate Cas9 proteins after completion of the intended editing, or that prevent Cas9 activity altogether at undesired times or in unintended tissues. We recently discovered natural Cas9 inhibitors [anti-CRISPR (Acr) proteins] that evolved as counter-measures against CRISPR immunity. We have identified and validated five distinct families of Acrs from bacterial species with Type II-C CRISPR-Cas systems, and established their efficacy as off-switches for NmeCas9 genome editing in human cells. Our proven strategies have also led us to additional Acr candidates that are likely inhibitors of distinct Type II-C Cas9 orthologs. Our discoveries therefore provide us with a powerful entrée into addressing the limitations and safety concerns that arise from uncontrolled or otherwise unwanted Cas9 activity. In this proposal, we outline experiments that exploit our discovery of Cas9 inhibitors in three ways. In Aim 1, we will use Acr phylogenetic distributions as pointers towards orthologous, robust, compact Type II-C Cas9 genome-editing systems, each of which would be immediately amenable to off-switch control. Because distinct Cas9s often have novel targeting specificities, this work also promises to expand the genomic scope of Cas9 editing. In Aim 2, we will optimize Acr inhibitory potency, and test the hypothesis that Acrs can be used to increase the efficiency of precise editing via homology-dependent repair. Finally, in Aim 3, we will establish tissue-specific Acr control over genome editing in cultured mammalian cells and use it to develop a flexible platform for restricting Cas9 genome editing to a single desired cell type. Our strategy for enforcing tissue specificity of gene editing will be applicable even to cell types in which tissue-specific promoters are unavailable for driving Cas9 expression. The proposed research promises to yield enhanced genome-editing systems with broader targeting range, fewer safety risks and improved tissue specificity, which we will demonstrate pre-clinically in mammalian cells.

修改项目摘要/摘要部分 CRISPR-Cas9通过其RNA引导的能力来靶向单个基因进行破坏或精确编辑，正在彻底改变生命科学。除了加速基础研究和生物技术之外，Cas9及其RNA指导还具有通过基于基因组编辑的治疗来改变遗传性疾病治疗的潜力，特别是如果可以克服交付障碍并证明临床安全性的话。Cas9系统有三种亚型（II-A、B和II-C型），并且所有三种都产生了在哺乳动物基因组编辑中具有证明的效用的Cas9直系同源物。我们自己的工作已经从N.脑膜炎病毒（NmeCas 9）作为一个紧凑的，自然超准确的基因组编辑平台。Cas9临床开发中的一些安全性问题源于Cas9活性过度、延长或存在于非预期的细胞类型中。由于缺乏有效的工具，这些问题已经加剧，这些工具可以在完成预期的编辑后抑制Cas9蛋白，或者在不希望的时间或在非预期的组织中完全阻止Cas9活性。 我们最近发现了天然的Cas9抑制剂[抗CRISPR（Acr）蛋白]，它们是针对CRISPR免疫的对抗措施。我们已经用II-C型CRISPR-Cas系统从细菌物种中鉴定并验证了五个不同的Acr家族，并确定了它们作为人类细胞中NmeCas 9基因组编辑的关闭开关的功效。我们经过验证的策略还使我们找到了其他Acr候选物，它们可能是不同II-C型Cas9直系同源物的抑制剂。因此，我们的发现为我们提供了一个强大的入口，以解决不受控制或不必要的Cas9活性所引起的限制和安全问题。 在这个提案中，我们概述了以三种方式利用我们发现的Cas9抑制剂的实验。在目标1中，我们将使用Acr系统发育分布作为指向正交的、稳健的、紧凑的II-C型Cas9基因组编辑系统的指针，其中每一个都将立即服从开关控制。由于不同的Cas9通常具有新的靶向特异性，这项工作也有望扩大Cas9编辑的基因组范围。在目标2中，我们将优化Acr抑制效力，并测试Acr可用于通过同源依赖性修复提高精确编辑效率的假设。最后，在目标3中，我们将在培养的哺乳动物细胞中建立组织特异性Acr对基因组编辑的控制，并使用它来开发一个灵活的平台，用于将Cas9基因组编辑限制在单一的所需细胞类型。我们实施基因编辑的组织特异性的策略甚至适用于组织特异性启动子无法驱动Cas9表达的细胞类型。 拟议的研究有望产生增强的基因组编辑系统，具有更广泛的靶向范围，更少的安全风险和更高的组织特异性，我们将在哺乳动物细胞中进行临床前证明。

项目成果

Shutting down RNA-targeting CRISPR.

关闭 RNA 靶向 CRISPR。

• DOI: 10.1126/science.abc8243
• 发表时间: 2020
• 期刊: Science (New York, N.Y.)
• 作者: Barrangou,Rodolphe;Sontheimer,ErikJ
• 通讯作者: Sontheimer,ErikJ

Adenine Base Editing In Vivo with a Single Adeno-Associated Virus Vector.

• DOI: 10.1089/genbio.2022.0015
• 发表时间: 2022-06
• 期刊: GEN biotechnology
• 作者: Han Zhang;Nathan Bamidele;Pengpeng Liu;O. Ojelabi;Xin D Gao;Tom S Rodriguez;Hao-Feng Cheng

Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo.

• DOI: 10.1038/s41467-021-26518-y
• 发表时间: 2021-11-01
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Ibraheim R;Tai PWL;Mir A;Javeed N;Wang J;Rodríguez TC;Namkung S;Nelson S;Khokhar ES;Mintzer E;Maitland S;Chen Z;Cao Y;Tsagkaraki E;Wolfe SA;Wang D;Pai AA;Xue W;Gao G;Sontheimer EJ
• 通讯作者: Sontheimer EJ

CRISPR Shields: Fending Off Diverse Cas Nucleases with Nucleus-like Structures.

CRISPR Shields：利用类核结构抵御多种 Cas 核酸酶。

• DOI: 10.1016/j.molcel.2020.02.015
• 发表时间: 2020
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Barrangou,Rodolphe;Sontheimer,ErikJ
• 通讯作者: Sontheimer,ErikJ

A Hyperthermophilic Phage Decoration Protein Suggests Common Evolutionary Origin with Herpesvirus Triplex Proteins and an Anti-CRISPR Protein.

超嗜热噬菌体装饰蛋白表明与疱疹病毒三链体蛋白和抗 CRISPR 蛋白具有共同的进化起源。

• DOI: 10.1016/j.str.2018.04.008
• 发表时间: 2018
• 期刊: Structure (London, England : 1993)
• 作者: Stone,NicholasP;Hilbert,BrendanJ;Hidalgo,Daniel;Halloran,KevinT;Lee,Jooyoung;Sontheimer,ErikJ;Kelch,BrianA
• 通讯作者: Kelch,BrianA