Engineering Novel Precision Genome Editing Tools

工程新型精密基因组编辑工具

• 批准号: 10472939
• 负责人: Audrone Lapinaite
• 金额: $ 119.91万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472939
• 关键词: Address; Biotechnology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cystic Fibrosis; DNA; Detection; Disease; Engineering; GTP-Binding Protein alpha Subunits, Gs; Gene Expression; Genetic Diseases; Genetic Variation; Genomics; Health; Human Genetics; Livestock; Molecular; Neighborhoods; Neurodegenerative Disorders; Nucleotides; Pathogenicity; Pathway interactions; Point Mutation; Progeria; Purines; Pyrimidines; RNA Editing; Sickle Cell Anemia; Single Nucleotide Polymorphism; Syndrome; Therapeutic; Therapeutic Agents; Work; base; base editor; cancer type; design; genome editing; in vivo; innovation; interest; next generation; novel; novel therapeutic intervention; nuclease; prime editor; programs; repaired; tool

PROJECT SUMMARY/ABSTRACT Half of the known human genetic variations that contribute to disease are due to single nucleotide polymorphisms (SNPs). Thus, there is a pressing need to develop precision genome editing tools that are able to correct these SNPs with high efficiency and accuracy. Current CRISPR-Cas based precision genome editing tools, such as DNA base editors and prime editors, were designed to perform targeted single nucleotide changes without introducing double stranded breaks and relying on the homology-directed repair pathway. However, these tools have several drawbacks observed in cells, such as off-target DNA and RNA editing, low efficiency, and unintended editing of nucleotides within the neighborhood of the target nucleotide (bystander editing) leading to undesired genomic changes. Moreover, DNA base editors are able to perform only transitions (interchanging purines (AG) or pyrimidines (CT)) but not transversions (interchanging pyrimidines for purines and vice versa). These shortcomings reduce the targeting capabilities of current precision genome editing tools and are the key limitations of using them as therapeutic agents. Building on our recent work that explains the molecular basis of the DNA base editors’ drawbacks, we propose four innovative strategies to design precision genome editing approaches that address the limitations of current genome editing tools and expand their targeting scope. Three of the four strategies will yield base editors with dual programmability. Besides the programable nuclease (Cas9) that guides base editors to the sequence of interest, these novel base editors will additionally have easily programmable catalytic modules that will allow selecting only one nucleotide for editing. This dual programmability will eliminate the bystander editing and make these DNA base editors exceptionally accurate. Two out of four designs will yield DNA base editors able to perform transversions (interchanging pyrimidines for purines (CG and TG)) and to correct additional ~25% of pathogenic SNPs inaccessible by current base editors. Moreover, one of the two transversion base editors will possess dual programmability, hence will be exceptionally accurate. Overall, the four strategies proposed here will yield the next generation precision genome editing tools that, besides the direct therapeutic corrections of SNPs’ in vivo, will also allow interrogating the association between multiple SNPs, gene expression and diseases (neurodegenerative diseases or various types of cancers). Thus, these DNA editing tools will pave the way for investigating the molecular mechanisms of multiple genetic disorders and enable us to develop new therapeutic strategies.

项目概要/摘要 导致疾病的已知人类遗传变异中有一半是由单核苷酸引起的 多态性（SNP）。因此，迫切需要开发能够实现精准基因组编辑的工具。 高效、准确地校正这些 SNP。当前基于 CRISPR-Cas 的精准基因组编辑 DNA 碱基编辑器和 Prime 编辑器等工具旨在执行有针对性的单核苷酸变化 无需引入双链断裂并依赖同源定向修复途径。然而， 这些工具在细胞中观察到了一些缺点，例如 DNA 和 RNA 编辑脱靶、效率低、 以及目标核苷酸附近的核苷酸的意外编辑（旁观者编辑）导致 不良的基因组变化。此外，DNA 碱基编辑器只能执行转换（交换 嘌呤（AG）或嘧啶（CT）），但不是颠换（将嘧啶互换为嘌呤，反之亦然） 反之亦然）。这些缺点降低了当前精准基因组编辑工具的靶向能力，并且 使用它们作为治疗剂的主要局限性。以我们最近解释分子的工作为基础 针对DNA碱基编辑器的缺点，我们提出了四种创新策略来设计精准基因组 解决当前基因组编辑工具的局限性并扩大其靶向范围的编辑方法。 四种策略中的三种将产生具有双重可编程性的碱基编辑器。除了可编程核酸酶 （Cas9）引导碱基编辑器找到感兴趣的序列，这些新颖的碱基编辑器还可以轻松地 可编程催化模块，允许仅选择一种核苷酸进行编辑。这个双 可编程性将消除旁观者编辑并使这些 DNA 碱基编辑器异常准确。 四分之二的设计将产生能够执行颠换的 DNA 碱基编辑器（将嘧啶互换为 嘌呤（CG 和 TG））并纠正当前碱基无法访问的额外约 25% 的致病性 SNP 编辑们。此外，两个颠换碱基编辑器之一将具有双重可编程性，因此将 异常准确。总的来说，这里提出的四种策略将产生下一代精准基因组 编辑工具，除了在体内对 SNP 进行直接治疗校正外，还可以询问 多个SNP、基因表达和疾病（神经退行性疾病或各种疾病）之间的关联 癌症类型）。因此，这些 DNA 编辑工具将为研究分子机制铺平道路 多种遗传性疾病的研究，使我们能够开发新的治疗策略。