Generation and Validation of a Novel Genome-Scale Inducible RNAi Library for Functional Genetics in Leishmania braziliensis.

用于巴西利什曼原虫功能遗传学的新型基因组规模诱导性 RNAi 文库的生成和验证。

• 批准号: 10726352
• 负责人: PHILLIP A YATES
• 金额: $ 23.1万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10726352
• 关键词: Acceleration; Bar Codes; Biology; CRISPR/Cas technology; Communities; Cutaneous Leishmaniasis; DNA Library; Defect; Disease; Double-Stranded RNA; Drug resistance; Ensure; Enterobacteria phage P1 Cre recombinase; Essential Genes; Generations; Genes; Genetic; Genetic Recombination; Genetic Screening; Genetic Transcription; Genome; Genomic DNA; Glean; Goals; Growth; Incidence; Individual; Leishmania; Leishmania braziliensis; Libraries; Life Cycle Stages; Messenger RNA; Methods; Miltefosine; Molecular Genetics; Mucocutaneous leishmaniasis; Parasites; Pathology; Pathway interactions; Peptides; Persons; Pharmaceutical Preparations; Pharmacotherapy; Plasmids; Production; Proteins; RNA; RNA Interference; RNA interference screen; Reproducibility; Resistance; Resources; Ribosomal RNA; Ribosomes; Sirolimus; Site; System; Tertiary Protein Structure; Transfection; Trypanosoma brucei brucei; Validation; Viannia; constitutive expression; drug action; fitness; genome-wide; human disease; improved; inducible Cre; inducible gene expression; innovation; insight; knock-down; loss of function; member; neglected tropical diseases; new therapeutic target; novel; novel therapeutics; recombinase; resistance gene; stem; tool; vector

Project Summary Leishmania parasites cause a suite of devastating Neglected Tropical Diseases that afflict as many as one million people per year. Genetic tools for understanding important questions in Leishmania biology are currently quite limited. Most Leishmania species lack the capacity for RNA interference (RNAi), precluding the implementation of genome-scale RNAi library screens like those that have revolutionized molecular genetics in the related kinetoplastid T. brucei. However, the Viannia subgenus, which includes Leishmania braziliensis, has retained the RNAi machinery. We have developed the first inducible RNAi system for L. braziliensis and have adapted it for high throughput Loss-of-Function RNAi screens. The overall goal of this proposal is to generate and validate a novel, genome-scale, inducible RNAi library in L. braziliensis that will serve as a versatile new genetic tool for the field. Despite differences in the capacity for RNAi, the genome organization, life cycle stages, and disease pathology of L. braziliensis are very similar to other Leishmania species, making it likely that results from L. braziliensis RNAi screens will be broadly applicable. Our inducible expression system is based on conditional site-specific recombination by a split-Cre recombinase (DiCre) that is only active in the presence of the inducer rapamycin (Rap). For inducible RNAi, Rap treatment activates DiCre to invert a pair of tandemly duplicated RNAi target sequences into an antiparallel orientation. Transcription of this inverted repeat generates a long double-stranded RNA stem-loop that serves as a trigger for RNAi knockdown of the targeted mRNA. Specific Aim 1 has three components: 1) We will generate a genome-scale plasmid library of random ~1 kb L. braziliensis genomic DNA fragments that will serve as RNAi targeting sequences. 2) This plasmid library will be converted to a barcoded inducible stem- loop RNAi library via a method developed in our lab called Strand Displacement Duplication (SDD); 3) The inducible stem-loop RNAi library will be stably transfected into a DiCre-expressing L. braziliensis strain, using CRISPR/Cas9 cleavage to improve library integration efficiency. Because the overall goal is to make the RNAi library available as a resource for the community, we will validate the library by performing proof-of-principle screens to identify drug resistance genes (Gain-of-Fitness) and pathways required for normal growth (Loss-of- Fitness) (Specific Aim2). The representation of RNAi target sequences in library under various growth conditions will be assessed via an adaptation of the barcode sequencing method (Bar-seq). The L. braziliensis RNAi library platform will provide an innovative and much-needed new tool for high throughput genetic screens in Leishmania. We anticipate that genome-scale RNAi screens will have a sustained impact on the field by revealing mechanisms of drug action and resistance, and uncovering proteins and pathways required for surviving any growth condition or lifecycle stage.

项目摘要 利什曼原虫引起一系列毁灭性的被忽视的热带病， 每年一百万人。用于理解利什曼原虫生物学中重要问题的遗传工具是 目前相当有限。大多数利什曼原虫物种缺乏RNA干扰（RNAi）的能力，排除了利什曼原虫的基因表达。 实施基因组规模的RNAi文库筛选，如那些已经彻底改变分子遗传学， 相关动质体T.布鲁塞。然而，Viannia亚属，包括巴西利什曼原虫， 保留了RNAi机制我们成功地构建了第一个针对L.巴西和 已经将其用于高通量的功能丧失RNAi筛选。本提案的总体目标是 在L.巴西，将作为一个 多功能的新基因工具。尽管RNAi的能力不同，基因组的组织， 生活史阶段和疾病病理学。巴西利什曼原虫与其他利什曼原虫非常相似， 这可能是由L.巴西RNA干扰筛选将是广泛适用的。 我们的诱导表达系统是基于条件性位点特异性重组的分裂Cre 重组酶（DiCre），其仅在诱导物雷帕霉素（Rap）存在下有活性。对于可诱导的RNAi， Rap处理激活DiCre以将一对串联重复的RNAi靶序列转化为一个串联重复的RNAi靶序列。 反平行取向这个反向重复序列的转录产生一个长的双链RNA茎环 其作为靶mRNA的RNAi敲低的触发物。具体目标1有三个组成部分： 我们将产生一个基因组规模的随机~1 kb L质粒文库。巴西人基因组DNA片段， 将作为RNA干扰靶向序列。2)该质粒文库将被转化为条形码化的诱导型茎- 通过我们实验室开发的称为链置换复制（SDD）的方法构建环状RNAi文库; 3） 诱导型茎环RNA干扰文库将稳定转染到表达DiCre的L.巴西菌株，使用 CRISPR/Cas9切割以提高文库整合效率。因为总的目标是让RNAi 图书馆可作为社区的资源，我们将通过执行原理证明来验证图书馆 筛选以确定耐药基因（适应性获得）和正常生长所需的途径（适应性丧失）。 具体目标2）。不同生长条件下RNAi靶序列在文库中的表达 条件将通过条形码测序方法（Bar-seq）的调整进行评估。 洛杉矶巴西RNAi文库平台将为高水平的基因工程提供创新和急需的新工具。 在利什曼原虫中进行基因筛查。我们预计，基因组规模的RNAi筛选将有一个 通过揭示药物作用和耐药性机制以及揭示蛋白质， 以及在任何生长条件或生命周期阶段生存所需的途径。