Shenzhen Key Laboratory of Gene Regulation and Systems Biology, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen 518055, China.
Computational Bioscience Research Center (CBRC), Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia.
Nucleic Acids Res. 2022 Mar 21;50(5):e26. doi: 10.1093/nar/gkac108.
Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3'UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.
可变聚腺苷酸化（APA）在基因调控中起着重要作用。随着新型测序技术在 APA 分析中的应用，越来越多的 APA 基因/位点被鉴定出来。然而，这些 APA 异构体的大多数表型相关性仍然难以捉摸，这主要是由于缺乏一种方便的 APA 干扰遗传学工具。为了解决这个问题，本文基于 CRISPR-dCas13 系统开发了一种有效的方法，称为 CRISPR-iPAS。在八种不同的 dCas13 蛋白中，牙龈卟啉单胞菌（Porphyromonas gulae，Pgu）dCas13b 被鉴定为最有效地阻断聚腺苷酸化位点（PAS）使用的蛋白。通过靶向核心调控元件的向导 RNA，dPguCas13b 能够调节具有不同 APA 类型的内源性基因，包括串联 3'UTR、交替末端外显子以及内含子 PAS。最后，我们证明了所提出的 APA 扰动工具可用于研究 APA 异构体的功能相关性。
Zotero 插件
