Uncovering the molecular function of novel transposon-encoded Cas9 homologs

揭示新型转座子编码的 Cas9 同源物的分子功能

• 批准号: 10609818
• 负责人: Chance Meers
• 金额: $ 7.18万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10609818
• 关键词: Adaptive Immune System; Address; Adopted; Affect; Antibiotic Resistance; Arginine; Bacteria; Binding; Biochemical; Biochemistry; Bioinformatics; Biological Assay; Biological Models; Biological Process; Clustered Regularly Interspaced Short Palindromic Repeats; Comparative Genomic Analysis; Complex; DNA; DNA Sequence; DNA Transposable Elements; Deoxyribonuclease I; Development; EMSA; Elements; Endowment; Enzymes; Escherichia coli; Event; Evolution; Excision; Family; Foundations; Frequencies; Future; Genes; Genetic; Genetic Engineering; Genetic Transcription; Genetic Variation; Genome; Genome engineering; Guide RNA; Homologous Gene; Homologous Protein; Horizontal Gene Transfer; Immune response; Immune system; In Vitro; Jaw; Life; Mediating; Mobile Genetic Elements; Modeling; Molecular; Monitor; Mutagenesis; Nature; Northern Blotting; Nucleic Acids; Open Reading Frames; Organism; Parasites; Partner in relationship; Process; Property; Protein Family; Proteins; RNA; RNA-Protein Interaction; Race; Rag1 Mouse; Role; Secondary to; Site; Specificity; Structure; System; Testing; Time; Training; Transcript; Transposase; Untranslated RNA; V(D)J Recombination; Vertebrates; Viral; Work; adaptive immunity; assault; design; driving force; endonuclease; experimental study; fitness; genetic association; genome editing; genome-wide; in vivo; insight; novel; nuclease; protein function; resistance gene; scaffold; tool; transposon sequencing; virulence gene; yeast two hybrid system

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.

项目摘要 移动遗传因素（MGE）具有动员基因组和/或之间的动员能力 来自不同物种的基因组，是抗生素耐药性和病毒传播的主要驱动力 基因。由于MGE的持续攻击，实质性生物已经进化出了许多复杂的 具有先天和自适应水平的防御系统，在某些情况下直接针对MGE 核酸以序列特异性方式。近年来，很重要的是免疫系统 由簇的定期间隔短的短质体重复序列（CRISPR）和CRISPR相关（CAS）编码（CAS） 基因，该基因指导RNA的靶向结合和外核酸的裂解。 值得注意的是，CAS基因本身是从MGES内编码的基因和转座子演变而来的 尤其。一个引人注目的例子是RNA引导的DNA核酸内切酶Cas9，它直接从一个 TNPB家族中不同的同源转座子编码蛋白（并提到） 作为CAS9H），其分子函数完全未知。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个构成的非编码RNA（NCRNA），该RNA显示出强大的遗传 与CAS9H基因的关联。我假设古老的转座子编码CAS9同源物功能 与类似指南的RNA一起调节满意度，效率和/或目标位点特异性 移动元件的换位。通过发现这种原始生物学功能，我的工作将提供 对CRISPR-CAS9的进化轨迹和发现自然使用的核心酶特性的见解 作为起始材料，可以获得潜在的，高度可编程的RNA引导的DNA核酸内切酶。 在AIM 1中，我将生物信息识别包含CAS9同源物的可转座元素以优先考虑 实验研究的候选人，并开发出强大的异源表达系统以监测 转置事件。重要的是，通过监视全基因组插入特异性，将告知CAS9H是否 调节目标选择。在AIM 2中，我将通过 系统的诱变，并确定cas9h核酸酶结构域在转座中的作用。最后，在AIM 3中 我将采用生化方法直接探测Cas9H和NCRNA之间的蛋白-RNA相互作用， 并揭示其组成的核酸酶结构域的作用，为将来的结构研究奠定了基础 可能会阐明TNPB和Cas9之间的古老脚手架。这个项目将利用我的培训 作为分子遗传学家，扩大了我在生物信息学和生物化学方面的能力。这些实验将是 第一个严格探测TNPB家族蛋白的功能，并首次揭示TNPB/CAS9H的功能 调节换位。除了提供有关CRISPR-CAS免疫进化起源的见解 系统，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。