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

项目总结 CRISPR相关转座子(CAST)代表了一类新发现的细菌基因 可以执行可编程换位的元素。使用用户提供的RNA序列，CAST可以 将千碱基的DNA插入基因组中的特定位置，这是一项无法通过 目前最先进的基因组编辑工具。因此，管型符合基因疗法中尚未得到满足的需求 受基因突变困扰的疾病，如囊性纤维化。为下一代应用优化投射 基因组编辑工具，需要全面了解它是如何插入DNA的。特别感兴趣的 是它的单向性，指的是CAST倾向于在一个方向上插入DNA。而当 单向性是CAST的一个令人垂涎的特征，其机制仍未确定。隐含的组件 促成这一机制的是元素两侧的签名端。这些末端招募蛋白质，TnsB 在模拟系统shCAST中，将转座子从其原始位置切除，以便为插入做准备 其他地方。然而，这些末端在顺序上是不同的，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，可以用来实现单向性。 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因素。 特殊目标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 配对末端复合体，涉及不对称末端之间的相互作用的转位阶段，由 TnsB。这种结构将从3D的角度识别将末端彼此区分开来的关键特征。 转座体的后续特征，转座的下一阶段涉及组装 与其他CAST蛋白配对的末端复合体，将识别识别的特征如何传达 单向性。特定目标2将利用生物信息学来识别所有演员组中保存的特征 建立定向专一性的系统。元基因组挖掘将扫描巨大的序列数据宝库，以 编制一份全面的演员阵容元素清单。此扩展集允许对类型转换进行进化分析 确定保持单向性的全球趋势，并将根据以下内容定义每个要素的机制 保留保留的特征。生化分析将用于验证在这两个目标中确定的特征。 PI将接受冷冻-EM和生物信息学技能以及批判性思维的广泛培训 确保实验和计算结果的无缝集成。凯洛格和费肖特实验室位于 康奈尔大学可以获得广泛的资源，以确保掌握这些技能和意志 还强调沟通等软技能，以确保PI发展为全面发展的科学家。 综上所述，这项建议提出了一项全面理解CAST单向性及其 相关机制，使其能够在感兴趣的基因组编辑工具中进行优化和实施。

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.