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

项目摘要 CRISPR相关转座子（CAST）代表了一类最近发现的细菌遗传修饰。 可以执行可编程换位的元件。使用用户提供的RNA序列，CAST可以 将DNA插入到基因组中的特定位点，这是一个关键的功能， 最先进的基因组编辑工具。因此，CAST匹配遗传性肿瘤治疗中未满足的需求。 比如囊性纤维化优化CAST以应用于下一代 基因组编辑工具，需要全面了解它如何插入DNA。特别感兴趣的 是它的单向性，指的是CAST的倾向，插入DNA在一个单一的方向。而 单向性是CAST的一个令人垂涎的特征，但其机制仍然没有被描述。隐含组件 元件侧面的标记末端有助于这种机制。这些末端募集蛋白质， 在模型系统shCAST中，将转座子从其原始位置切除以准备插入 其他地方然而，这些末端在序列上不同，在本提案中被描述为“不对称”， 这表明TnsB在每一端执行不同的功能，可以用于实现单向性。 该项目提出了一个跨学科项目，将确定单向性的关键驱动因素。 具体目标1将使用高分辨率冷冻电子显微镜（cryo-EM）对shCAST进行结构可视化 双末端复合物，涉及不对称末端之间相互作用的转座阶段， TnsB.该结构将从3D角度识别将两端彼此区分开的关键特征。 转座体的后续表征，转座的下一阶段涉及转座体的组装 与其他CAST蛋白质的配对末端复合物，将识别所识别的特征如何传达 单向性具体目标2将利用生物信息学来识别所有CAST中保守的特征 建立定向特异性的系统。宏基因组挖掘将扫描大量的序列数据， 编制一份全面的CAST元素列表。这个扩展的集合使得能够对CAST进行进化分析， 确定保持单向性的全球趋势，并将根据 保留保守的特征。将使用生化测定来验证在任一目标中鉴定的特征。 PI将接受冷冻EM和生物信息学技能的广泛培训，沿着批判性思维， 确保实验和计算结果的无缝集成。凯洛格和费肖特实验室在 康奈尔大学有机会获得广泛的资源，以确保掌握这些技能和意志 还强调软技能，如沟通，以确保PI的发展，作为一个全面的科学家。 总之，该提案提出了一个全面了解CAST单向性及其 相关的机制，使其能够在感兴趣的基因组编辑工具中优化和实现。

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.