A massively parallel reporter assay for measuring chromatin effects on alternative splicing

用于测量染色质对选择性剪接的影响的大规模并行报告分析

• 批准号: 9977420
• 负责人: Georg Seelig
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-11 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977420
• 关键词: 3' Untranslated Regions; Alternative Splicing; Bar Codes; Behavior; Biological Assay; Buffers; CRISPR library; CRISPR/Cas technology; Chromatin; Code; Consumption; DNA; DNA Methylation; DNA Modification Process; DNA cassette; Data; Digestion; Elements; Epigenetic Process; Episome; Exons; Frequencies; Future; Gene Expression; Genes; Genetic Transcription; Genetic Variation; Genome; Genome Mappings; Genomic DNA; Genomics; Guide RNA; Human; Human Genetics; Inverse Polymerase Chain Reaction; Learning; Libraries; Location; Maps; Measurement; Measures; Messenger RNA; Modeling; Nonhomologous DNA End Joining; Nucleotides; Pathway interactions; Polyadenylation; Positioning Attribute; Protein Isoforms; Protocols documentation; RNA; RNA Splicing; RNA library; Regulation; Regulatory Element; Reporter; Reporter Genes; Research Project Grants; Resolution; Running; Signal Transduction; Site; Technology; Testing; Time; Tissues; Transcript; Transgenes; Variant; Work; base; computerized tools; design; endonuclease; experimental study; gene therapy; genome editing; genome-wide; genomic locus; histone modification; improved; integration site; new technology; novel; predictive modeling; predictive tools; promoter; rapid technique; recruit; role model; technology development; tool; transcriptome sequencing; whole genome

Tools for mapping transgene insertion sites and associated expression levels are broadly useful but current approaches are experimentally cumbersome and have limited throughput. Here, we propose to develop a novel type of reporter construct, genome reporter interprocessing (GRiP), that makes it dramatically easier to randomly integrate reporter genes in a large number of genomic locations and subsequently map the insertion site. We will apply GRiP technology to integrate a library of alternatively spliced reporter constructs into hundreds of thousands of different positions, thus enabling us to understand how alternative splice isoform ratios vary between different genomic contexts. We will use the resulting data to improve existing computational tools for predicting the impact of variants on alternative splicing. GRiP builds on existing technologies and pathways, most importantly CRISPR/Cas9 genome editing and transcript cleavage and polyadenylation (CPA). We will use a guide RNA (gRNA) library and Cas9 endonuclease activity to create double stranded breaks in a large but defined set of genomic locations. A linear reporter cassette will be co-transfected with the gRNA library and, at some frequency, will be ligated into the break through the non-homologous end joining pathway. The reporter cassette consists of, at least, a promoter, coding sequence and a truncated 3’UTR that ends directly at the core signal recruiting the CPA machinery. To perform insertion mapping, we take advantage of a key feature of CPA, namely the ~17 nucleotide (nt) distance between the core signal and the position of transcript cleavage and polyadenylation. Because the core signal of the reporter cassette will be directly ligated to genomic DNA, transcription will run through the end of the cassette and cleavage will occur ~17 nt into the neighboring genomic sequence. As a result, each transcript’s GRiP site will carry a ~17 nt “barcode” that reveals information about the site of integration. By sequencing the reporter transcripts and by combining the barcode information with information about the possible insertion sites (as determined by the chosen gRNAs), both location of insertion and transcriptional activity can be precisely mapped. While we here focus on initial technology development and on applying GRiP to investigate alternative splicing in the genome, we expect that this technology will find a wide range of additional applications including the precise mapping of double-stranded breaks generated during genome editing.

用于映射转换插入站点和相关表达式水平的工具广泛有用，但当前 方法在实验上很麻烦，吞吐量有限。在这里，我们建议开发小说 记者构造的类型，基因组记者解释（抓地力），使其动态更容易随机随机 在大量基因组位置中的集成报告基因，然后绘制插入位点。我们 将应用抓地力技术将剪接的记者结构的库集成到数百个库中 成千上万的不同位置，从而使我们能够了解替代剪接同工型比率如何变化 在不同的基因组环境之间。我们将使用结果数据来改善现有的计算工具 预测变体对替代剪接的影响。抓地力基于现有技术和途径， 最重要的是CRISPR/CAS9基因组编辑和转录本裂解和聚腺苷酸化（CPA）。我们将使用 引导RNA（GRNA）库和Cas9核酸内切酶活性，以在大型但 定义的一组基因组位置。线性记者盒将与GRNA库共转染，并在 一些频率将连接到突破穿过非理论端连接途径的突破。记者 纸盒至少由启动子，编码顺序和直接在核心结束的截断的3'UTR组成 信号招募CPA机械。要执行插入映射，我们利用CPA的关键功能 即核心信号与转录本裂解位置之间的〜17个核苷酸（NT）距离 聚腺苷酸化。因为记者盒的核心信号将直接连接到基因组DNA，所以 转录将通过盒式末端运行，裂解将发生〜17 nt进入相邻的基因组。 顺序。结果，每个成绩单的抓地点将携带约17 nt的“条形码”，以揭示有关的信息 集成站点。通过对报告者的成绩单进行排序，并将条形码信息与 有关可能的插入位点的信息（由选定的GRNA确定），这是插入的两个位置 可以精确映射转录活性。当我们在这里专注于初始技术开发和 在应用握把研究基因组中的替代剪接时，我们希望这项技术会发现广泛 其他应用程序范围包括在此期间生成的双链断裂的精确映射 基因组编辑。