(27am) An Ultrasensitive Amplification-Free Nucleic Acid Detection Using CRISPR Chain Reaction v2

The CRISPR/Cas-based detection systems are widely used for molecular diagnostics and are known for detecting nucleic acids accurately and rapidly. Recently, several groups, including our lab, introduced various amplification-free technologies based on combining multiple CRISPR/Cas systems for detecting genomic DNA with attomolar sensitivity including CONAN¹, FIND-IT², CRISPR Chain Reaction v1 (CCR-1)³. However, the translation of these methods is limited by low signal-to-noise ratio and long duration for sample-to-result. To overcome these limitations, we have further engineered an ultrasensitive CRISPR Chain Reaction v2 (CCR-2) by combining a primary active CRISPR/Cas system with another inactive secondary CRISPR/Cas system consisting of an excess synthetic DNA and locked crRNA.

Here, we have designed >20 types of blockers that hybridize to either the scaffold, spacer, or enhance region of the crRNAs to investigate the "locked" crRNAs that work efficiently for this system. These designs were created by varying the positions of a non-complementary ‘loop’ region that can be cleaved via trans-cleavage activity of the primary CRISPR/Cas system. In the presence of a target, the primary CRISPR/Cas system gets activated and cleaves the loops on secondary crRNAs, unlocking it. The activated secondary system rapidly unlocks inactive secondary system, which results in an exponential amplification signal, in contrast to the linear signal with traditional Cas12a-based methods. Therefore, with the CCR-2, we can amplify the signal instead of amplifying DNA. We have multiplexed various CRISPR/Cas systems for primary and secondary system to enhance the sensitivity of detection. CCR-2 offers a simple and rapid next-generation test for detecting nucleic acids and can potentially be adopted for low-resource settings.

References

1. Shi K, Xie S, Tian R, Wang S, Lu Q, Gao D, Lei C, Zhu H, Nie Z. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci Adv. 2021 Jan 27;7(5):eabc7802. doi: 10.1126/sciadv.abc7802. PMID: 33571114; PMCID: PMC7840123.
2. Liu, T.Y., Knott, G.J., Smock, D.C.J. et al.Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol17, 982–988 (2021). https://doi.org/10.1038/s41589-021-00842-2
3. Rananaware, S. et al. Amplification-free nucleic acid detection at room temperature using CRISPR chain reaction. In: 2021 AlChE Annual Meeting; Nov 7-11, 2021; Boston, MA. Abstract 160x.