Programmable RNA detection with CRISPR-Cas12a

Abstract

CRISPR is a prominent bioengineering tool and the type V CRISPR-associated protein complex, Cas12a, is widely used in diagnostic platforms due to its innate ability to cleave DNA substrates. Here we demonstrate that Cas12a can also be programmed to directly detect RNA substrates without the need for reverse transcription or strand displacement. We discovered that while the PAM-proximal seed region of the crRNA exclusively recognizes DNA for initiating trans-cleavage, the PAM-distal region or 3'-end of the crRNA can tolerate both RNA and DNA substrates. Utilizing this property, we developed a method named Split Activators for Highly Accessible RNA Analysis (SAHARA) to detect RNA sequences at the PAM-distal region of the crRNA by merely supplying a short ssDNA or a PAM containing dsDNA to the seed region. Notably, SAHARA is Mg2+ concentration- and pH-dependent, and it was observed to work robustly at room temperature with multiple orthologs of Cas12a. SAHARA also displayed a significant improvement in the specificity for target recognition as compared to the wild-type CRISPR-Cas12a, at certain positions along the crRNA. By employing SAHARA we achieved amplification-free detection of picomolar concentrations of miRNA-155 and hepatitis C virus RNA. Finally, SAHARA can use a PAM-proximal DNA as a switch to control the trans-cleavage activity of Cas12a for the detection of both DNA and RNA targets. With this, multicomplex arrays can be made to detect distinct DNA and RNA targets with pooled crRNA/Cas12a complexes. 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.

A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA

“…When detecting RNA by Cas12a, reverse transcription and subsequent amplification reactions are required since the Cas12a enzyme does not naturally tolerate RNA substrates. Recently, Jain et al developed a split-activator-based method called SAHARA (19) for programmable RNA detection with Cas12a. However, their diagnostic method has two major limitations: (i) only picomolar levels (250-700 pM) of RNA detection was obtained which does not meet the needs of clinical testing, and (ii) the specificity in more complex samples is not good enough since the method is designed to only bind to 12-nt of the target RNA.…”

“…Recently, Jain et al developed a diagnostic method that, for the first time, enables direct detection of RNA by Cas12a (19). By supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA, the method detected RNA substrates at the 3’-end of the crRNA.…”

A split crRNA with CRISPR-Cas12a enables highly sensitive, selective, and multiplexed detection of RNA and DNA

“…When detecting RNA by Cas12a, reverse transcription and subsequent ampli cation reactions are required since the Cas12a enzyme does not naturally tolerate RNA substrates. Recently, Jain et al developed a split-activator-based method called SAHARA (19) for programmable RNA detection with Cas12a.…”

“…Recently, Jain et al developed a diagnostic method that, for the rst time, enables direct detection of RNA by Cas12a (19). By supplying a short ssDNA or a PAM-containing dsDNA at the seed region of the crRNA, the method detected RNA substrates at the 3'-end of the crRNA.…”

A Ratiometric Nonfluorescent CRISPR Assay Utilizing Cas12a-Induced Plasmid Supercoil Relaxation

“…Numerous CRISPR-Dx platforms have been developed to date, showcasing the immense potential of CRISPR systems in addressing the longstanding challenge of achieving highly sensitive and specific nucleic acid detection through a comparatively straightforward process 14,15, . CRISPR-Cas12a particularly draws extensive attention, given its ease of reconfiguration for detecting a wide range of human, animal, and plant diseases 12,19,22,25,[49][50][51][52] , including gene mutations 34,53 .…”