Developing programmable RNA writing tools with the novel RNA-guided RNA-targeting CRISPR effector Cas7-11

使用新型 RNA 引导的 RNA 靶向 CRISPR 效应器 Cas7-11 开发可编程 RNA 写入工具

• 批准号: 10736989
• 负责人: Omar O Abudayyeh
• 金额: $ 55.65万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-23 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736989
• 关键词: Acceleration; Binding; Biochemical; Bioinformatics; Biology; Biomedical Research; Cell model; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Viruses; Development; Disease; Disease model; Dose; Engineering; Enzymes; Excision; Exons; Family; Genes; Genetic Diseases; Genome; Guide RNA; Human; Individual; Location; Methods; Modality; Modification; Molecular Biology; Neurodegenerative Disorders; Neurologic; Neurons; Nucleic Acids; Orthologous Gene; Patients; Performance; Point Mutation; Process; Protein Engineering; Proteins; RNA; RNA Editing; RNA Sequences; RNA Splicing; Reaction; Research Personnel; Resources; Safety; Specificity; System; Technology; Therapeutic; Tissues; Toxic effect; Trans-Splicing; Transcript; Translations; Trinucleotide Repeats; Work; Writing; base; base editing; causal variant; cell type; genome editing; improved; induced pluripotent stem cell; insight; knock-down; mRNA Precursor; neuropsychiatric disorder; novel; novel therapeutics; nuclease; recruit; structural biology; success; technology development; therapeutic RNA; therapeutically effective; tool

Project Summary: While gene editing technologies have revolutionized the ability to programmably edit DNA with high efficiency in diverse tissues, there remain several challenges with DNA editing, including permanent off-targets, concern for permanent correction of certain diseases, and some diseases being better targeted by other modalities than gene editing. For example, treatment of triplet repeat disorders with gene editing remains difficult, due to the difficulty of targeting repeat regions in the genome and the need to make large and precise deletions, without causing off-target genome rearrangements and other undesired effects on the genome. RNA modifications, however, may offer a better approach with notable features: 1) temporal and reversible modification of genetic diseases, 2) minimal off-targets which are reversible and less harmful, and 3) more versatile editing beyond genome editing. For example, with triplet repeat disorders, an RNA writing strategy could allow for collapse of the repeats to the exact desired number, an approach that would be more successful than gene editing or RNA knockdown strategies that have failed. To accomplish RNA writing, which involves all possible base edits (transitions and transversions), small or large insertions, and small or large replacements (e.g. exon swapping), some approaches have been developed, such as trans-splicing, but with limited success. Trans-splicing relies on the recruitment of an RNA template to a pre-mRNA without any active targeting domains and involves competition with the cis target. As a result, programmable trans-splicing has had low efficiency. We hypothesized that combining trans-splicing with programmable RNA guided CRISPR systems could help boost the efficiency of the trans-splicing mechanism, enabling any potential type of RNA edit, insertion, deletion, or replacement to be incorporated into endogenous transcripts. While we and others have characterized novel programmable RNA targeting CRISPR systems, such as Cas13, and developed tools from these systems, use of these tools have been limited in cellular systems due to a non-promiscuous cleavage activity known as collateral activity. While Cas13 has been shown to have specific RNA cleavage activity in some cell types, other cell types have had significant collateral cleavage of cellular RNAs, leading to toxicity in cell models. The proposed work will address these needs by combining biochemical characterization, structural characterization, and enzyme engineering to develop new RNA targeting CRISPR nucleases without collateral activity, such as the novel CRISPR-Cas7-11 enzyme, for specific RNA writing tools in conjunction with trans-splicing to enable any possible RNA edit. Beyond optimizing the RNA writing technology via trans-splicing optimization using RNA and protein engineering, we will showcase RNA writing’s therapeutic potential by correcting triplet repeat disorders in iPSC-derived human neurons. The developed technologies in this proposal will accelerate the pace of biomedical research and enable treatment of many genetic disorders, many of which are not treatable with gene editing, bringing more therapies to patients.

项目摘要：虽然基因编辑技术彻底改变了编辑DNA的能力 由于多样性时机的效率很高，DNA编辑仍然存在一些挑战，包括永久性 脱离目标，关注某些疾病的永久纠正以及某些疾病的目标是 除基因编辑以外的其他方式。例如，使用基因编辑的三重症重复疾病处理仍然存在 由于难以靶向基因组中的重复区域的困难，并且需要大量而精确 删除，不会引起脱靶基因组重排和对基因组的其他不希望的影响。 RNA 但是，修改可能提供更好的方法，具有显着特征：1）临时和可逆 遗传疾病的修饰，2）最小的脱靶是可逆且有害的，3）更多 超越基因组编辑的多功能编辑。例如，有了三重重复疾病，RNA写作策略 可以使重复崩溃到确切的所需数字，这种方法将更多 比失败的基因编辑或RNA敲低策略成功。完成RNA写作，这 涉及所有可能的基础编辑（过渡和横向），小或大插入以及小或大的 替代品（例如外显子交换），已经开发了一些方法，例如变形，但有 有限的成功。跨拼接依赖于募集RNA模板到没有任何活性的前MRNA 定位域并涉及与CIS目标的竞争。结果，可编程的移媒体具有 效率低。我们假设将变速箱与可编程的RNA指导CRISCR相结合 系统可以帮助提高移媒体机制的效率，从而实现任何潜在类型的RNA 编辑，插入，删除或替换，将其纳入内源成绩单。当我们和其他人 已经表征了新颖的可编程RNA靶向CRISPR系统，例如CAS13，并开发了工具 从这些系统中，由于非公认为 裂解活性称为附带活性。虽然已证明CAS13具有特定的RNA裂解 在某些细胞类型中的活性，其他细胞类型的细胞RNA具有明显的附带裂解，导致 细胞模型中的毒性。拟议的工作将通过结合生化特征来满足这些需求， 结构表征和酶工程，以开发针对CRISPR核的新RNA 没有附带活动，例如新颖的CRISPR-CAS7-11酶，用于特定的RNA编写工具 与移序连接以启用任何可能的RNA编辑。除了优化RNA写作技术之外 通过使用RNA和蛋白质工程进行的跨式切割优化，我们将展示RNA写作的治疗性 通过纠正IPSC衍生的人类神经元中的三胞胎重复疾病的潜力。发达的技术 该建议将加速生物医学研究的速度，并能够治疗许多遗传疾病， 其中许多无法通过基因编辑来治疗，从而为患者带来更多疗法。