Circular RNA as a platform for genome-wide microRNA sponge libraries

环状 RNA 作为全基因组 microRNA 海绵文库的平台

• 批准号: 10009531
• 负责人: Brian Frederick Pickering
• 金额: $ 22.5万
• 依托单位: CHIMERNA THERAPEUTICS INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-03 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009531
• 关键词: 3' Untranslated Regions; Bar Codes; Binding; Binding Sites; Biomedical Research; Catalytic RNA; Cell physiology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Cytosol; Data; Disease; Elements; Family; Gene Expression; Gene Expression Profiling; Genetic Transcription; Genome; Genomics; Goals; Impairment; Individual; Introns; Legal patent; Libraries; Mediating; Messenger RNA; MicroRNAs; Mutate; Output; Pathway interactions; Phase; Physiological; Porifera; Problem Solving; Process; Proteins; RNA; RNA Degradation; RNA Ligase (ATP); RNA Sequences; RNA-Binding Proteins; Regulator Genes; Resistance; Role; Scientist; Seeds; Site; System; Technology; Testing; Time; Tornadoes; Transcript; Universities; circular RNA; experimental study; genome wide screen; genome-wide; mRNA Transcript Degradation; member; new technology; novel; novel strategies; screening; small hairpin RNA

SUMMARY: MicroRNAs are small (19 to 22-nt long) RNAs that are major regulators of gene expression. MicroRNAs bind to specific mRNAs to promote their degradation. A major goal is to discover microRNAs that contribute to cellular and disease processes. The ability to discover microRNAs involved in cellular pathways using unbiased genomic screening is highly limited. In contrast to mRNAs, microRNAs cannot be easily screened in a genome-wide manner for their role in diverse processes using bar-coded lentiviral CRISPR and shRNA libraries. Here we propose a fundamentally new approach that will lead to a highly efficient library for screening all microRNAs encoded by the genome. Our approach is to express a library of circular RNAs that act as “microRNA sponges,” each tailored to sequester a specific microRNA in the genome. Although circular RNAs are known to be effective microRNA sponges, the inability to express circular RNAs at high levels in cells has limited their ability to sponge microRNAs, which are highly abundant. Chimerna scientists, while working at Cornell University, developed a fundamentally new strategy to express circular RNA at 100X higher levels than any other previous expression system. Because of this powerful and recently developed circular RNA expression system, we can now generate libraries of circular RNA sponges that can systematically sponge each microRNA in the cell. Chimerna scientists have generated key poof-of-concept data demonstrating the efficacy of this approach with a circular RNA that sponges microRNA miR-19. In order to develop and optimize microRNA sponges to make them suitable for creating a genome-wide lentiviral library, the specific aims of this proposal are (1) To optimize circular RNA sponges to achieve maximal depletion of microRNA activity in cells. In this subaim, we will optimize the sponging activity of circular RNA sponges by testing the optimal spacing and the number of microRNA-binding sites. Additionally, microRNA sponges will be developed that can sponge multiple co-regulated microRNAs. Overall, these experiments will lead to optimized RNA circles that are highly effective in sponging one or more microRNA seed families. (2) To systematically test and reduce off-target effects of circular microRNA sponges. In this aim we will optimize circular RNA sponges by reducing any off-target effects. We will use a gene expression analysis approach to quantify the degree of on-target and off-target effects induced by the circular RNA sponges. To remove any off-target effects, we will systematically mutate sequences that may contribute to these effects. Together, these experiments will result in circular RNAs that have reduced off target effects and will therefore be more suitable for developing a microRNA-sponging library. Overall, this project will result in optimized circular RNA sponges with maximal sponging activity and reduced off-target effects on gene expression. The resulting circles, which will be expressed in a lentiviral system, will be suitable for the next phase of this project, which will involve construction of a genome-wide library, targeting over 2000 microRNAs.

摘要：microRNAs是一种小的(19到22个核苷酸长)RNA，是基因表达的主要调节因子。 MicroRNAs与特定的mRNAs结合以促进其降解。一个主要目标是发现能够 有助于细胞和疾病过程。发现参与细胞通路的microRNAs的能力 使用无偏见的基因组筛选是非常有限的。与mRNAs不同，microRNAs不容易 以全基因组的方式使用条码慢病毒CRISPR和 ShRNA文库。在这里，我们提出了一种全新的方法，它将产生一个高效的库 筛选基因组编码的所有microRNA。我们的方法是表达一个环状RNA文库， 充当“微RNA海绵”，每个海绵都是为隔离基因组中的特定微RNA而量身定做的。虽然是圆形的 已知RNA是有效的microRNA海绵，无法在体内高水平表达环状RNA 细胞限制了它们吸收高度丰富的microRNA的能力。Chimerna科学家，而 在康奈尔大学工作，他开发了一种全新的策略，以100倍的速度表达循环RNA 比以往任何一种表达系统都要高。因为这个强大的新近开发的环状 RNA表达系统，我们现在可以产生环状RNA海绵库，它可以系统地 用海绵吸去细胞中的每个微小RNA。Chimerna科学家已经产生了关键的概念缺失数据 用海绵microRNA miR-19的环状RNA证明了这种方法的有效性。为了 开发和优化microRNA海绵，使其适合创建全基因组慢病毒文库， 该方案的具体目标是(1)优化环状RNA海绵以实现最大耗竭 细胞中微小RNA活性的变化。在这个子目标中，我们将优化环状RNA海绵的海绵活性 通过测试最优间距和microRNA结合位点的数量。此外，微RNA海绵将 可以结合多个共同调节的microRNA的海绵。总体而言，这些实验将导致 优化的RNA环，在海绵吸收一个或多个microRNA种子家族方面非常有效。(2)至 系统地测试和减少环形微RNA海绵的脱靶效应。为了实现这一目标，我们将 通过减少任何偏离目标的影响来优化环形RNA海绵。我们将使用基因表达分析 量化圆形RNA海绵引起的靶上和靶外效应程度的方法。至 去除任何偏离目标的影响，我们将系统地突变可能导致这些影响的序列。 总而言之，这些实验将产生环状RNA，减少了靶标效应，因此 更适合于开发microRNA海绵文库。总体而言，该项目将导致优化 环状RNA海绵具有最大的海绵活性，并减少了对基因表达的非靶向影响。这个 产生的圆圈将以慢病毒系统表达，将适用于该项目的下一阶段， 这将涉及建立一个全基因组文库，目标是2000多个microRNA。