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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要移动遗传元件（MGE）具有在基因组内和/或基因组之间移动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由于MGE的无情攻击，原核生物进化出了许多复杂的基因。在先天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 近年来，核酸以序列特异性方式变得尤为重要。由成簇规则间隔的短回文重复序列 (CRISPR) 和 CRISPR 相关序列 (Cas) 编码基因，利用引导 RNA 来靶向结合和切割外源核酸。值得注意的是，Cas 基因本身是从 MGE 和转座子中编码的基因进化而来的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 DNA 进化而来。 TnpB 家族中一组不同的同源转座子编码蛋白（以下称为 Cas9H），除了这些最初的生物信息学观察之外，其分子功能尚不清楚，我最近的分析发现了一种保守的非编码 RNA (ncRNA)，它显示出很强的遗传性我热衷于古老的转座子编码的 Cas9 同源物的功能。与引导样 RNA 一起，调节切除过程中的切除、效率和/或靶位点特异性通过发现这种原始的生物功能，我的工作将提供移动元素的换位。深入了解 CRISPR-Cas9 的进化轨迹并揭示大自然使用的核心酶特性作为起始材料来获得有效且高度可编程的 RNA 引导的 DNA 核酸内切酶。在目标 1 中，我将通过生物信息学方法识别包含 Cas9 同源物的转座元件，以进行优先级排序实验研究的候选者，并开发强大的异源表达系统来监测重要的是，通过监测全基因组插入特异性将判断 Cas9H 是否存在。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 TnpB 和 Cas9 之间保存的古代支架，该项目将利用我的训练。作为一名分子遗传学家，并扩展我在生物信息学和生物化学方面的能力。第一个严格探究TnpB家族蛋白的功能，并首次揭示TnpB/Cas9H如何除了提供对 CRISPR-Cas 免疫进化起源的见解之外。系统，这项工作的完成也将为新的基因工程工具的开发提供信息。