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可编程编辑 DNA 的能力 虽然 DNA 编辑在不同组织中具有高效率，但仍然存在一些挑战，包括永久性的 脱靶，关注永​​久纠正某些疾病，以及某些疾病可以更好地针对某些疾病 基因编辑以外的其他方式仍然存在，例如，通过基因编辑治疗三联体重复疾病。 困难，因为定位基因组中的重复区域很困难，并且需要制造大而精确的 删除，不会导致脱靶基因组重排和对基因组的其他不良影响。 然而，修改可能会提供一种更好的方法，具有显着的特点：1）时间和可逆 遗传疾病的修饰，2）最小的脱靶，可逆且危害较小，3）更多 基因组编辑之外的多功能编辑，例如，针对三联体重复紊乱，RNA 写入策略。 可以允许将重复次数折叠到所需的确切数量，这种方法会更有效 比基因编辑或 RNA 敲除策略更成功的方法是完成 RNA 写入。 涉及所有可能的碱基编辑（转换和颠换）、小或大插入以及小或大 替代（例如外显子交换），已经开发了一些方法，例如反式剪接，但 反式剪接的成功程度有限，依赖于将 RNA 模板招募到没有任何活性的前体 mRNA 上。 靶向域并涉及与顺式靶标的竞争，因此，可编程反式拼接已成为可能。 我们发现将反式剪接与可编程 RNA 引导 CRISPR 相结合的效率很低。 系统可以帮助提高反式剪接机制的效率，使任何潜在类型的RNA成为可能 当我们和其他人进行编辑、插入、删除或替换时，将其合并到内源转录本中。 表征了新型可编程 RNA 靶向 CRISPR 系统（例如 Cas13），并开发了工具 在这些系统中，由于非混杂性，这些工具的使用在蜂窝系统中受到限制 Cas13 已被证明具有特定的 RNA 切割活性。 某些细胞类型的活性，其他细胞类型对细胞 RNA 具有显着的附带裂解，导致 细胞模型中的毒性。拟议的工作将通过结合生化表征来满足这些需求， 结构表征和酶工程开发新的 RNA 靶向 CRISPR 核酸酶 没有附带活性，例如新型 CRISPR-Cas7-11 酶，用于特定 RNA 写入工具 与反式剪接结合，除了优化 RNA 写入技术之外，还可以实现任何可能的 RNA 编辑。 通过使用 RNA 和蛋白质工程进行反式剪接优化，我们将展示 RNA 写入的治疗作用 通过纠正 iPSC 衍生的人类神经元中的三联体重复紊乱来发挥潜力。 该提案将加快生物医学研究的步伐，并使许多遗传性疾病的治疗成为可能， 其中许多无法通过基因编辑来治疗，从而为患者带来更多治疗方法。