反式激活crRNA
清脆的
核糖核酸
DNA
计算生物学
生物
核酸
寡核苷酸
遗传学
Cas9
基因
分子生物学
作者
Santosh R. Rananaware,Emma K. Vesco,Grace M. Shoemaker,Swapnil S. Anekar,Luke Samuel W. Sandoval,Katelyn S. Meister,Nicolas C. Macaluso,Long Thanh Nguyen,Piyush Jain
标识
DOI:10.1038/s41467-023-41006-1
摘要
Abstract Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.
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