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Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a
Analytical Chemistry ( IF 7.4 ) Pub Date : 2023-05-30 , DOI: 10.1021/acs.analchem.3c00414
Qian Li 1 , Zhi-Ling Song 1 , Yuxi Zhang 1 , Lina Zhu 1 , Qian Yang 1 , Xingfu Liu 1 , Xufeng Sun 1 , Xuxu Chen 1 , Rongmei Kong 2 , Gao-Chao Fan 1 , Xiliang Luo 1
Affiliation  

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a “synergistic activator effect” is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the “helper” activator. The finding of “synergistic activator effect” not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

中文翻译:

两种 ssDNA 激活剂的协同掺入增强了 CRISPR/Cas12a 的反式切割

由于其放大的反式切割特征,CRISPR/Cas12a 被认为在分子检测和诊断方面具有强大的功能。然而,Cas12a 系统的激活特异性和多重激活机制尚未完全阐明。在此,发现了一种“协同激活剂效应”,它支持一种激活机制,即两个短 ssDNA 激活剂的协同掺入可以促进 CRISPR/Cas12a 的反式切割,而它们中的任何一个都太短而不能独立工作。作为概念验证示例,协同激活剂触发的 CRISPR/Cas12a 系统已成功用于 AND 逻辑运算和单核苷酸变异的鉴别,不需要信号转换元件或其他扩增酶。而且,通过预先引入 crRNA 和“辅助”激活剂之间的合成错配,实现了单核苷酸特异性检测单核苷酸变异。“协同激活效应”的发现不仅提供了对 CRISPR/Cas12a 更深入的了解,而且可能促进其扩展应用,并推动探索其他 CRISPR/Cas 系统未被发现的特性。
更新日期:2023-05-30
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