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bioRxiv · 10.1101/2023.01.29.525716

Programmable RNA detection with CRISPR-Cas12a

Rananaware, S. R.·Vesco, E. K.·Shoemaker, G. M.·Anekar, S. S.·Sandoval, L. S. W.·Meister, K. S.·Macaluso, N. C.·Nguyen, L. T.·Jain, P. K.
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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 or 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. O_FIG O_LINKSMALLFIG WIDTH=125 HEIGHT=200 SRC="FIGDIR/small/525716v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@5edb2dorg.highwire.dtl.DTLVardef@187678borg.highwire.dtl.DTLVardef@9f0184org.highwire.dtl.DTLVardef@11cb1a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Rananaware, S. R., Vesco, E. K., Shoemaker, G. M., Anekar, S. S., Sandoval, L. S. W., Meister, K. S., Macaluso, N. C., Nguyen, L. T., Jain, P. K.. 2023-01-30. Programmable RNA detection with CRISPR-Cas12a. https://doi.org/10.1101/2023.01.29.525716

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