Uncovering the molecular function of novel transposon-encoded Cas9 homologs

项目总结 移动遗传元件(MGE)具有在基因组内和/或在基因组之间进行动员的能力 来自不同物种的基因组，是抗生素耐药性和毒力传播的主要驱动力 基因。由于MGES的无情攻击，原核生物进化出了无数复杂的 同时在固有和适应性水平上运行的防御系统，在某些情况下直接针对MGE 核酸以序列特定的方式。近年来值得注意的是免疫系统。 由簇状规则间隔短回文重复(CRISPR)和CRISPR相关(CA)编码 利用引导RNA进行外源核酸的定向结合和切割的基因。 值得注意的是，CAS基因本身是从MGES和转座子中编码的基因进化而来的 尤其是。一个明显的例子是RNA引导的DNA内切酶Cas9，它直接从一个 TnpB家族中一组不同的同源转座子编码蛋白(以下称为 如Cas9H)，其分子功能完全未知。除了这些最初的生物信息学观察之外， 我最近的分析发现了一种保守的非编码RNA(NcRNA)，它显示出强大的遗传 与Cas9H基因的相关性。我推测古老的转座子编码的Cas9同源基因功能 与类似向导的RNA一起，调节切除、效率和/或靶点特异性 移动元件的移位。通过发现这种原始的生物学功能，我的工作将提供 洞察CRISPR-Cas9的进化轨迹并揭示大自然使用的核心酶特性 作为获得有效且高度可编程的RNA引导的DNA内切酶的起始材料。 在目标1中，我将通过生物信息识别包含Cas9同系物的转座元件，以确定优先顺序 作为实验研究的候选者，并开发一个强大的异源表达系统来监控 换位事件。重要的是，通过监测全基因组插入的特异性将告知Cas9H 调整目标选择。在目标2中，我将通过以下方式阐明非编码‘HEARO’RNA的功能 系统诱变，并确定Cas9H核酸酶结构域在转座中的作用。最后，在目标3中， 我将采用生化方法直接探测Cas9H和ncRNA之间的蛋白质-RNA相互作用， 并揭示其保守的核酸酶结构域的作用，为未来的结构研究奠定基础 可能会揭示保存在TnpB和Cas9之间的古代脚手架。这个项目将利用我的培训 作为一名分子遗传学家，并扩大我在生物信息学和生物化学方面的能力。这些实验将是 首次严格探索TnpB家族蛋白的功能，并首次揭示TnpB/Cas9H是如何 调节转位。除了提供对CRISPR-Cas免疫进化起源的见解 这项工作的完成还将为新的基因工程工具的开发提供信息。

PROJECT SUMMARY Mobile genetic elements (MGEs) possess the ability to mobilize within genomes and/or between genomes from distinct species, and are a major driving force in the spread of antibiotic resistance and virulence genes. Due to the unrelenting assault of MGEs, prokaryotic organisms have evolved numerous sophisticated defense systems that operate on both an innate and adaptive level, and in some cases directly target MGE nucleic acids in a sequence-specific manner. Of notable importance in recent years are immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, which employ guide RNAs for the targeted binding and cleavage of foreign nucleic acids. Remarkably, Cas genes themselves have evolved from genes encoded within MGEs, and transposons in particular. A striking example is the RNA-guided DNA endonuclease, Cas9, which evolved directly from a distinct group of homologous transposon-encoded proteins within the TnpB family (and is hereafter referred to as Cas9H), whose molecular functions are entirely unknown. Beyond these initial bioinformatic observations, my recent analyses have uncovered a conserved non-coding RNA (ncRNA) that shows strong genetic association with Cas9H genes. I hypothesize that ancient, transposon-encoded Cas9 homologs function together with guide-like RNAs, to modulate the excision, efficiency, and/or target site specificity during transposition of the mobile element. By discovering this primordial biological function, my work will offer insights into the evolutionary trajectory of CRISPR-Cas9 and uncover core enzymatic properties that nature used as starting material to arrive at a potent and highly programmable RNA-guided DNA endonuclease. In Aim 1, I will bioinformatically identify transposable elements containing Cas9 homologs to prioritize candidates for experimental study, and develop a robust heterologous expression system to monitor transposition events. Importantly by monitoring genome-wide insertion specificity will inform whether Cas9H modulates target selection. In Aim 2, I will elucidate the function of the non-coding ‘HEARO’ RNA through systematic mutagenesis, and determine the role of Cas9H nuclease domains in transposition. Finally, in Aim 3, I will adopt a biochemical approach to directly probe protein-RNA interactions between Cas9H and the ncRNA, and uncover the role of its conserved nuclease domains, laying a foundation for future structural studies that may shed light on ancient scaffolds conserved between TnpB and Cas9. This project will leverage my training as a molecular geneticist and expand my abilities in bioinformatics and biochemistry. These experiments will be the first to rigorously probe the function of a TnpB family protein, and reveal for the first time how TnpB/Cas9H modulates transposition. In addition to providing insights into the evolutionary origins of CRISPR-Cas immune systems, completion of this work will also inform the development of new genetic engineering tools.