在本地序列上下文中对近端和远端拼接调节元件进行深入选
Yocelyn Recinos1, Dmytro Ustianenko1,2, Yow-Tyng Yeh1
1Department of Systems Biology, Department of Biochemistry and Molecular Biophysics, Center for Motor Neuron Biology and Disease, Columbia University, New York, NY 10032, USA.
bioRxiv : the preprint server for biology
|September 4, 2023
概括
研究人员开发了基于CRISPR的选工具SpliceRUSH,用于在基因中映射拼接调节元件 (SREs). 这种方法在SMN2基因中确定了新型SREs,推动了针对脊髓肌肉缩等疾病的基于RNA的药物发现.
科学领域:
- 分子生物学分子生物学
- 遗传学 遗传学 是一个
- 在RNA生物学,RNA生物学.
背景情况:
- 前传递 RNA (前mRNA) 拼接对于基因表达至关重要,可以作为治疗干预的目标.
- 在它们的原生序列环境中识别剪接调节元件 (SREs) 是具有挑战性的,阻碍了向疗法的开发.
- 反感性寡核酸 (ASOs) 是调节拼接的一种模式,但目标识别仍然是一个瓶.
研究的目的:
- 开发一种高通量选方法,用于系统地绘制SREs.
- 利用可编程的RNA向系统,通过干扰内生拼接因子来调节拼接.
- 应用这项技术来识别用于治疗向的新型SRE,以SMN2基因为例.
主要方法:
- 开发SpliceRUSH,一种使用催化死CRISPR-RfxCas13dRNA向系统 (dCas13d/gRNA) 的高通量选方法.
- 一个lentivirus gRNA库的设计,将遗传区域划分,包括远端内基序列,以映射SREs.
- 将SpliceRUSH应用于SMN2基因,这是脊柱肌肉缩的治疗标.
主要成果:
- 在SMN2基因中,SpliceRUSH成功地绘制了SREs的地图,识别了已知的和一种新的远端内拼接增强剂.
- 该dCas13d/gRNA系统通过与内生拼接因子竞争,有效调节拼接.
- 准已识别的新增强剂改变了SMN2.2中的第7外子拼接.
结论:
- SpliceRUSH提供了一个强大的平台,可以在任何感兴趣的基因中系统地映射SRE.
- 这项技术加深了对拼接调节及其调节的理解.
- 这些发现对基于RNA的药物发现和对遗传疾病的新疗法开发有重大影响.
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