Structural and evolutionary basis for insertion unidirectionality in RNA-guided DNA transposition systems

RNA引导的DNA转座系统中插入单向性的结构和进化基础

• 批准号: 10752287
• 负责人: Vinh H Truong
• 金额: $ 4.77万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-16 至 2026-11-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10752287
• 关键词: 3-Dimensional; Affinity; Architecture; Bacteria; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Biological Models; CRISPR-associated transposons; Classification; Communication; Complex; Conserved Sequence; Critical Thinking; Cryoelectron Microscopy; Cystic Fibrosis; DNA; DNA Transposable Elements; DNA Transposons; Data; Development; Disease; EMSA; Elements; Ensure; Exhibits; Foundations; Gene Delivery; Gene Mutation; Genes; Genetic; Genetic Diseases; Genome; Guide RNA; Interdisciplinary Study; Learning; Length; Location; Machine Learning; Metagenomics; Mining; Mobile Genetic Elements; Molecular; Molecular Cloning; Molecular Conformation; Mutagenesis; Positioning Attribute; Property; Proteins; RNA Sequences; Regulation; Resolution; Resources; Scanning; Scientist; Site; Specificity; Structure; System; Therapeutic; Training; Transposase; Universities; Variant; Visualization; bacterial genetics; combat; computational pipelines; follow-up; gene therapy; genetic element; genome editing; insight; integration site; interest; next generation; novel; recruit; skills; structural biology; tool; trend; 3-Dimensional; Affinity; Architecture; Bacteria; Binding; Binding Sites; Biochemical; Bioinformatics; Biological Assay; Biological Models; CRISPR-associated transposons; Classification; Communication; Complex; Conserved Sequence; Critical Thinking; Cryoelectron Microscopy; Cystic Fibrosis; DNA; DNA Transposable Elements; DNA Transposons; Data; Development; Disease; EMSA; Elements; Ensure; Exhibits; Foundations; Gene Delivery; Gene Mutation; Genes; Genetic; Genetic Diseases; Genome; Guide RNA; Interdisciplinary Study; Learning; Length; Location; Machine Learning; Metagenomics; Mining; Mobile Genetic Elements; Molecular; Molecular Cloning; Molecular Conformation; Mutagenesis; Positioning Attribute; Property; Proteins; RNA Sequences; Regulation; Resolution; Resources; Scanning; Scientist; Site; Specificity; Structure; System; Therapeutic; Training; Transposase; Universities; Variant; Visualization; bacterial genetics; combat; computational pipelines; follow-up; gene therapy; genetic element; genome editing; insight; integration site; interest; next generation; novel; recruit; skills; structural biology; tool; trend

PROJECT SUMMARY CRISPR-associated transposons (CAST) represent a class of recently discovered bacterial genetic elements that can perform programmable transposition. Using a user-provided RNA sequence, CASTs can insert kilobases of DNA into specific sites in the genome, a crucial function that cannot be achieved with current state of the art genome editing tools. Thus, CASTs match an unmet need in therapeutics for genetic disorders plagued by gene mutations like cystic fibrosis. To optimize CASTs for application in next-generation genome editing tools, a comprehensive understanding of how it inserts DNA is required. Of particular interest is its unidirectionality, referring to CAST’s propensity to insert DNA in a single orientation. While unidirectionality is a coveted feature of CASTs, its mechanism remains uncharacterized. Components implied to contribute to this mechanism are the signature ends flanking the element. These ends recruit proteins, TnsB in model system shCAST, that excise the transposon from its original location to prepare it for insertion elsewhere. However, these ends are different in sequence, described in this proposal as “asymmetric”, suggesting that TnsB performs different functions at each end that could be used to achieve unidirectionality. This project proposes an interdisciplinary project that will identify key drivers for unidirectionality. Specific aim 1 will use high-resolution cryo-electron microscopy (cryo-EM) to structurally visualize the shCAST paired-end complex, a stage of transposition involving interactions between the asymmetric ends facilitated by TnsB. This structure will identify key features that distinguish the ends from one another from a 3-D standpoint. Follow-up characterization of the transpososome, the next stage of transposition involving the assembly of the paired-end complex with other CAST proteins, will recognize how the identified features convey unidirectionality. Specific aim 2 will utilize bioinformatics to identify features conserved across all CAST systems that establish orientation specificity. Metagenomic mining will scan the vast trove of sequence data to compile a comprehensive list of CAST elements. This expansive set enables evolutionary analysis of CASTs to determine global trends maintaining unidirectionality and will define each element’s mechanism based on retention of conserved features. Biochemical assays will be used to validate features identified in either aim. The PI will be extensively trained in both cryo-EM and bioinformatics skills along with critical thinking to ensure seamless integration of experimental and computational results. The Kellogg and Feschotte labs at Cornell University have access to a wide-ranging set of resources that ensure mastery of these skills and will also emphasize soft skills like communication to ensure the PI’s development as a well-rounded scientist. Together, this proposal sets forth a plan to comprehensively understand CAST unidirectionality and its associated mechanisms, enabling its optimization and implementation in genome editing tools of interest.

项目摘要 CRISPR相关的转座子（CAST）代表了一类最近发现的细菌遗传 可以执行可编程换位的元素。使用用户提供的RNA序列，铸件可以 将DNA的千目标插入基因组中的特定位点，这是一种至关重要的功能，无法通过 当前的艺术基因组编辑工具。那是铸造与通用治疗中未满足的需求 受到囊性纤维化等基因突变困扰的疾病。优化用于下一代应用的演员 基因组编辑工具，对它如何插入DNA的全面了解。特别感兴趣 是它的单向性，指的是Cast在单一方向上插入DNA的承诺。尽管 单向性是铸造的令人垂涎的特征，其机制仍然没有表征。组件暗示 为此机制做出贡献是元素侧面的签名末端。这些末端募集蛋白质，TNSB 在模型系统shcast中，将转座子从其原始位置进行切除以准备插入 别处。然而，这些末端的顺序不同，在本提案中描述为“不对称”， 提示TNSB在两端执行不同的功能，可用于实现单向性。 该项目提出了一个跨学科项目，该项目将确定单向性的关键驱动力。 特定的目标1将使用高分辨率的低分子 - 电子显微镜（Cryo-EM）来结构可视化shcast 配对末端复合物，一个转位阶段，涉及由不对称末端之间相互作用的阶段 TNSB。该结构将确定将末端从3-D的角度区分开的关键特征。 转座体的后续表征，涉及组装的下一个转位阶段 配对 - 末端复合物与其他铸造蛋白将认识到确定的特征如何传达 单向性。特定的目标2将利用生物信息学来识别所有演员的保守特征 建立定向特异性的系统。宏基因组采矿将扫描序列数据的大量trove to 汇总铸造元素的综合列表。此额外的集合可以使演员表的进化分析 确定维持单向性的全球趋势，并将根据每个元素的机制定义 保留构成特征。生化测定将用于验证两个目标中识别的特征。 PI将接受Cryo-EM和生物信息学技能的广泛培训，以及批判性思维 确保实验和计算结果的无缝集成。 Kellogg和Feschotte实验室 康奈尔大学可以使用一套广泛的资源，以确保掌握这些技能并将 还强调了诸如沟通之类的软技能，以确保PI作为全面的科学家的发展。 该提议共同制定了一项计划，以全面了解铸造单向性及 相关机制，使其在感兴趣的基因组编辑工具中进行优化和实施。