Abstract
AbstractCas12a, 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.
Publisher
Springer Science and Business Media LLC
Subject
General Physics and Astronomy,General Biochemistry, Genetics and Molecular Biology,General Chemistry,Multidisciplinary
Reference52 articles.
1. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
2. Brouns Stan, J. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
3. Fineran, P. C. & Charpentier, E. Memory of viral infections by CRISPR-Cas adaptive immune systems: Acquisition of new information. Spec. Issue Viruses Microbes 434, 202–209 (2012).
4. Makarova, K. S. et al. Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).
5. Pinilla-Redondo, R. et al. Type IV CRISPR–Cas systems are highly diverse and involved in competition between plasmids. Nucleic Acids Res. 48, 2000–2012 (2020).
Cited by
36 articles.
订阅此论文施引文献
订阅此论文施引文献，注册后可以免费订阅5篇论文的施引文献，订阅后可以查看论文全部施引文献