美国普林斯顿大学Cameron Myhrvold课题组提出了RNA结构调节Cas13活性并使错配检测成为可能。这一研究成果发表在2025年10月23日出版的国际学术期刊《自然—生物技术》上。

课题组系统地探讨了二级结构对Cas13的影响。研究人员发现原间隔物中的二级结构及其3′抑制Cas13活性，并通过链位移框架定量解释了前者的作用。然后，研究小组利用链位移产生一个“闭塞”的Cas13，它将错配辨别提高了50倍，并能够在低（<1%）等位基因频率下识别序列不确定的突变。利用封闭的Cas13，课题组人员鉴定了SARS-CoV-2、人和甲型禽流感病毒主题中的人类适应性突变，以及KRAS中的致癌突变。他们的工作利用改进的对Cas13的机制理解来扩大RNA诊断的范围，并使结构知情的Cas13方法成为可能。

据悉，Cas13是通过CRISPR RNA与靶RNA中的互补单链RNA原间隔器杂交而激活的。虽然Cas13在体外不被双链RNA激活，但在RNA高度结构化的细胞环境中，它会自动靶向RNA。Cas13靶向结构化rna的机制尚不清楚。

附：英文原文

Title: RNA structure modulates Cas13 activity and enables mismatch detection

Author: Larsen, Benjamin B., Kimchi, Ofer, Dunkley, Owen R. S., Grimm, Maaike S., Siegers, Jurre Y., Huang, Yujia, Vandavasi, Venu G., Nguyen, Long T., Lamb, Caitlin H., Aranovich, Irina, Eggink, Dirk, Meijer, Adam, Jalal, Hamid, Notterman, Daniel A., Karlsson, Erik A., te Velthuis, Aartjan J. W., Myhrvold, Cameron

Issue&Volume: 2025-10-23

Abstract: Cas13 is activated by the hybridization of a CRISPR RNA to a complementary single-stranded RNA protospacer in a target RNA. While Cas13 is not activated by double-stranded RNA in vitro, it robustly targets RNA in cellular environments where RNAs are highly structured. The mechanism by which Cas13 targets structured RNAs remains unknown. Here, we systematically probe the effects of secondary structure on Cas13. We find that secondary structure in the protospacer and 3′ to it inhibits Cas13 activity and quantitatively explains the former effect through a strand displacement framework. We then harness strand displacement to generate an ‘occluded’ Cas13, which enhances mismatch discrimination up to 50-fold and enables sequence-agnostic mutation identification at low (<1%) allele frequencies. Using occluded Cas13, we identify human-adaptive mutations in SARS-CoV-2 and human and avian influenza A viruses, as well as oncogenic mutations in KRAS. Our work leverages improved mechanistic understanding of Cas13 to expand the scope of RNA diagnostics and enable structure-informed Cas13 approaches.

DOI: 10.1038/s41587-025-02868-6

Source: https://www.nature.com/articles/s41587-025-02868-6