Department of Microbiology, Biochemistry, and Molecular Genetics, Center for Cell Signaling, Rutgers New Jersey Medical School, Newark, NJ, United States.
Department of Microbiology, Biochemistry, and Molecular Genetics, Center for Cell Signaling, Rutgers New Jersey Medical School, Newark, NJ, United States; Program in Gene Expression and Regulation, Center for Systems and Computational Biology, The Wistar Institute, Philadelphia, PA, United States.
Methods Enzymol. 2021;655:459-482. doi: 10.1016/bs.mie.2021.04.016. Epub 2021 Jun 4.
The CRISPR/Cas9 technology is revolutionizing genomic engineering. The high efficiency and selectivity of the system have inspired the development of various derived tools for gene regulation at different levels, such as transcriptional activation or inhibition, epigenetic modification, splicing, and base editing. Cleavage and polyadenylation (CPA) is an essential 3' end maturation step for almost all eukaryotic mRNAs. CPA is tightly coupled with transcriptional termination, and its activity impacts gene expression. Over half of all human genes display alternative polyadenylation (APA), where multiple cleavage and polyadenylation sites (PASs) lead to mRNA isoforms with variable termini. APA isoforms often have distinct metabolisms, and their relative abundance can change drastically in different cells. Here, we describe a method based on delivering a catalytically dead Cas9 (dCas9) to genomic regions nears the PAS, which alters APA site usage in 3'UTRs or introns. This method, named CRISPRpas, allows investigators to examine functional significance of APA isoforms of individual genes. We also describe using the bioinformatics program APAlyzer to examine APA events of interest with RNA-seq data.
CRISPR/Cas9 技术正在引发基因组工程的革命。该系统的高效率和选择性激发了各种衍生工具的发展，这些工具可在不同水平上进行基因调控，如转录激活或抑制、表观遗传修饰、剪接和碱基编辑。切割和多聚腺苷酸化 (CPA) 是几乎所有真核 mRNA 的必需 3' 端成熟步骤。CPA 与转录终止紧密偶联，其活性影响基因表达。超过一半的人类基因显示出可变多聚腺苷酸化 (APA)，其中多个切割和多聚腺苷酸化位点 (PAS) 导致具有可变末端的 mRNA 异构体。APA 异构体通常具有不同的代谢物，其相对丰度在不同细胞中会发生剧烈变化。在这里，我们描述了一种基于将无催化活性 Cas9 (dCas9) 递送至 PAS 附近基因组区域的方法，该方法改变了 3'UTR 或内含子中的 APA 位点使用。这种方法被称为 CRISPRpas，允许研究人员检查单个基因的 APA 异构体的功能意义。我们还描述了使用生物信息学程序 APAlyzer 来检查具有 RNA-seq 数据的感兴趣的 APA 事件。
