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A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2018-02-02 00:00:00 , DOI: 10.1021/acschembio.7b00899
Tara R deBoer 1 , Noreen Wauford 1 , Jing-Yi Chung 1 , Miguel Salvador Torres Perez 1 , Niren Murthy 1
Affiliation  

The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alternative guide RNA validation system called GUIDER. GUIDER features a hairpin loop structure with a proximal guanosine-rich unit, a distal fluorophore unit, and a gRNA-targeting stem component. Cleavage of GUIDER by its complementary RNA-guided Cas9 endonuclease complex yields a fluorescent emission at 525 nm, signaling effective cleavage of the hairpin structure. GUIDER was validated using the model gene target mpcsk9, and it was able to identify the gRNA that could most efficiently cleave the target mpcsk9 gene. The modular design of GUIDER should allow it to have broad applicability in validating gRNAs, and its fluorescent signal output offers a rapid, simple, and quantitative measure of Cas9-mediated DNA cleavage.

中文翻译:


用于测定引导 RNA 活性的切割响应性茎环发夹



CRISPR-Cas9 技术的范围现在远远超出了基因组工程的范围。虽然人们付出了巨大的努力来推动这一革命性生物医学工具的发展,但体外裂解测定仍然是验证引导核酸内切酶 Cas9 到达所需基因组靶点的向导 RNA 的标准方法。在这里,我们报告了一种名为 GUIDER 的替代向导 RNA 验证系统的开发。 GUIDER 具有发夹环结构,具有近端富含鸟苷的单元、远端荧光团单元和 gRNA 靶向茎成分。 GUIDER 通过其互补的 RNA 引导的 Cas9 核酸内切酶复合物进行切割,产生 525 nm 的荧光发射,表明发夹结构的有效切割。 GUIDER 使用模型基因靶标mpcsk9进行了验证,并且能够识别能够最有效地切割靶标mpcsk9基因的 gRNA。 GUIDER 的模块化设计应使其在验证 gRNA 方面具有广泛的适用性,并且其荧光信号输出为 Cas9 介导的 DNA 切割提供了快速、简单和定量的测量。
更新日期:2018-02-02
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