Institut de Génétique Humaine, Université de Montpellier, CNRS UMR9002, Montpellier, France.
Institut Curie, Paris-Saclay Research University, CNRS UMR3348, 91401 Orsay, France.
Nucleic Acids Res. 2024 Oct 28;52(19):11926-11939. doi: 10.1093/nar/gkae682.
Alternative splicing allows multiple transcripts to be generated from the same gene to diversify the protein repertoire and gain new functions despite a limited coding genome. It can impact a wide spectrum of biological processes, including disease. However, its significance has long been underestimated due to limitations in dissecting the precise role of each splicing isoform in a physiological context. Furthermore, identifying key regulatory elements to correct deleterious splicing isoforms has proven equally challenging, increasing the difficulty of tackling the role of alternative splicing in cell biology. In this work, we take advantage of dCasRx, a catalytically inactive RNA targeting CRISPR-dCas13 ortholog, to efficiently switch alternative splicing patterns of endogenous transcripts without affecting overall gene expression levels cost-effectively. Additionally, we demonstrate a new application for the dCasRx splice-editing system to identify key regulatory RNA elements of specific splicing events. With this approach, we are expanding the RNA toolkit to better understand the regulatory mechanisms underlying alternative splicing and its physiological impact in various biological processes, including pathological conditions.
可变剪接允许从同一个基因产生多个转录本，从而在有限的编码基因组中多样化蛋白质组，并获得新的功能。它可以影响广泛的生物学过程，包括疾病。然而，由于在生理环境中精确解析每个剪接异构体的精确作用方面存在局限性，其意义长期以来一直被低估。此外，确定关键的调节元件以纠正有害的剪接异构体同样具有挑战性，这增加了在细胞生物学中解决可变剪接作用的难度。在这项工作中，我们利用 dCasRx，一种无催化活性的 RNA 靶向 CRISPR-dCas13 同源物，以高效地切换内源性转录本的可变剪接模式，而不会影响整体基因表达水平，具有成本效益。此外，我们展示了 dCasRx 剪接编辑系统在识别特定剪接事件的关键调节 RNA 元件方面的新应用。通过这种方法，我们正在扩展 RNA 工具包，以更好地理解可变剪接及其在各种生物学过程（包括病理条件）中的生理影响的调节机制。
