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

用于绘制转基因插入位置和相关表达水平的工具广泛有用，但目前 这些方法在实验上比较繁琐，而且吞吐量有限。在这里，我们建议开发一部小说 一种报告构建类型，基因组报告互处理(GRIP)，这使得随机选择 整合大量基因组位置中的报告基因，并随后定位插入位点。我们 将应用GRIP技术将交替拼接的报告器构建库集成到数百个 成千上万个不同的位置，从而使我们能够了解不同的剪接异构体比率是如何变化的 在不同的基因组环境中。我们将使用生成的数据来改进现有的计算工具 预测变异对选择性剪接的影响。GRIP建立在现有技术和途径的基础上， 最重要的是，CRISPR/Cas9基因组编辑、转录本切割和多聚腺苷化(CPA)。我们将使用 引导RNA(GRNA)文库和Cas9内切酶活性在大型但 已定义的基因组位置集。一个线性报告盒将与gRNA文库共转染，并在 在一定的频率下，会通过连接成断裂的非同源末端连接途径。这位记者 盒由至少一个启动子、编码序列和直接在核心结束的截短的3‘非编码区组成 招收注册会计师机器的信号。为了执行插入映射，我们利用了CPA的一个关键特性， 即核心信号和转录本切割位置之间的~17个核苷酸(NT)距离和 多聚腺苷酸化。因为报告盒的核心信号将直接连接到基因组DNA， 转录将贯穿盒式磁带的末端，并发生~17个核苷酸的裂解进入邻近的基因组 序列。因此，每一份成绩单的GRIP站点将带有一个~17新台币的条形码，该条形码显示以下信息 整合的场所。通过对记者抄本进行排序并将条形码信息与 关于可能的插入位置的信息(由所选的gRNA确定)，插入的位置 转录活动可以被精确地描绘出来。虽然我们在这里关注的是初始技术开发和 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找到广泛的 一系列其他应用，包括精确定位在以下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.