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

用于定位转基因插入位点和相关表达水平的工具是广泛有用的，但目前 这些方法在实验上是麻烦的并且具有有限的生产量。在这里，我们建议开发一种新的 一种报告基因构建体，基因组报告基因间处理（GRiP），这使得随机 将报告基因整合到大量基因组位置中，随后绘制插入位点。我们 将应用GRiP技术将一个可变剪接的报告基因构建体库整合到数百个 数千个不同的位置，从而使我们能够了解选择性剪接异构体比例如何变化 在不同的基因组背景下。我们将使用由此产生的数据来改进现有的计算工具， 预测变异体对可变剪接的影响。GRiP以现有技术和途径为基础， 最重要的是CRISPR/Cas9基因组编辑和转录物切割和聚腺苷酸化（CPA）。我们将使用 引导RNA（gRNA）文库和Cas9内切核酸酶活性，以在大的但 基因组位置的定义。线性报告盒将与gRNA文库共转染，并且在转染前， 一些频率，将被连接到突破通过非同源末端连接途径。记者 盒至少由启动子、编码序列和直接终止于核心的截短的3 'UTR组成 信号招募注册会计师机器。为了执行插入映射，我们利用CPA的一个关键特性， 即核心信号和转录物切割位置之间的~17个核苷酸（nt）的距离， 聚腺苷酸化因为报告盒的核心信号将直接连接到基因组DNA， 转录将贯穿盒的末端，并且切割将发生到邻近基因组的~17 nt处。 顺序因此，每个转录本的GRiP位点将携带一个约17 nt的“条形码”，该条形码揭示了以下信息： 融合的场所。通过对报告基因转录本进行测序并将条形码信息与 有关可能插入位点的信息（由所选gRNA确定），两个插入位置 和转录活性可以被精确映射。虽然我们在这里专注于初始技术开发， 在应用GRiP研究基因组中的选择性剪接时，我们预计这项技术将发现广泛的 一系列额外的应用，包括精确映射过程中产生的双链断裂 基因组编辑

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.