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：虽然基因编辑技术已经彻底改变了可编程编辑DNA的能力 由于在不同组织中的高效率，DNA编辑仍然存在几个挑战，包括永久性的 目标外，对某些疾病的永久纠正的担忧，以及一些疾病更好地被 基因编辑以外的其他方式。例如，用基因编辑技术治疗三联体重复疾病 困难，因为很难定位基因组中的重复区域，而且需要制作大而精确的 基因缺失，不会引起偏离目标的基因组重排和其他对基因组的不良影响。核糖核酸 然而，修改可能提供具有显著特征的更好的方法：1)时间性和可逆性 遗传疾病的修饰，2)可逆且危害较小的最小偏离目标，以及3)更多 除了基因组编辑外，还有多种编辑功能。例如，对于三联重复紊乱，一种RNA写入策略 可以将重复次数压缩到所需的确切数量，这一方法将比 成功的比基因编辑或RNA击倒策略失败的要好。为了完成RNA的写入，这 涉及所有可能的基本编辑(过渡和转换)、小插页或大插页以及小插页或大插页 替换(例如外显子交换)，已经开发了一些方法，例如反式剪接，但与 有限的成功。反式剪接依赖于在没有任何活性的情况下将RNA模板招募到前mRNA 以域名为目标，并涉及与独联体目标的竞争。因此，可编程的反式剪接具有 效率很低。我们假设，结合反式剪接和可编程RNA引导的CRISPR 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 编辑、插入、删除或替换，以并入内源性转录本。当我们和其他人 表征了针对CRISPR系统的新型可编程RNA，如Cas13，并开发了工具 在这些系统中，由于非混杂，这些工具在蜂窝系统中的使用已受到限制 卵裂活动称为附随活动。而Cas13已被证明具有特定的RNA切割作用 在某些类型的细胞中，其他类型的细胞有显著的侧枝裂解细胞RNA，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， 结构表征和开发针对CRISPR核酸酶的新RNA的酶工程 没有辅助活性，如新的CRISPR-Cas7-11酶，用于特定的RNA书写工具 与反式剪接相结合，实现任何可能的RNA编辑。超越优化的RNA写入技术 通过使用RNA和蛋白质工程的反式剪接优化，我们将展示RNA写作的治疗作用 通过纠正IPSC来源的人类神经元中的三联体重复障碍的潜力。中开发的技术 这项提议将加快生物医学研究的步伐，并使许多遗传疾病的治疗成为可能， 其中许多疾病无法通过基因编辑进行治疗，从而为患者带来了更多的治疗方法。