Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two Higher Eukaryotes and Prokaryotes Nucleotide (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.

An aerosol jet printing-enabled dual-function biosensor for the sensitive detection of pathogens using SARS-CoV-2 RNA as an example has been developed. A CRISPR-Cas13:guide-RNA complex is activated in the presence of a target RNA, leading to the collateral trans-cleavage of ssRNA probes that contain a horseradish peroxidase (HRP) tag. This, in turn, catalyzes the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by HRP, resulting in a color change and electrochemical signal change. The colorimetric and electrochemical sensing protocol does not require complicated target amplification and probe immobilization and exhibits a detection sensitivity in the femtomolar range. Additionally, our biosensor demonstrates a wide dynamic range of 5 orders of magnitude. This low-cost aerosol inkjet printing technique allows for an amplification-free and integrated dual-function biosensor platform, which operates at physiological temperature and is designed for simple, rapid, and accurate point-of-care (POC) diagnostics in either low-resource settings or hospitals.

A novel localized surface plasmon resonance (LSPR) system based on the coupling of gold nanomushrooms (AuNMs) and gold nanoparticles (AuNPs) is developed to enable a significant plasmonic resonant shift. The AuNP size, surface chemistry, and concentration are characterized to maximize the LSPR effect. A 31 nm redshift is achieved when the AuNMs are saturated by the AuNPs. This giant redshift also increases the full width of the spectrum and is explained by the 3D finite-difference time-domain (FDTD) calculation. In addition, this LSPR substrate is packaged in a microfluidic cell and integrated with a CRISPR-Cas13a RNA detection assay for the detection of the SARS-CoV-2 RNA targets. Once activated by the target, the AuNPs are cleaved from linker probes and randomly deposited on the AuNM substrate, demonstrating a large redshift. The novel LSPR chip using AuNP as an indicator is simple, specific, isothermal, and label-free; and thus, provides a new opportunity to achieve the next generation multiplexing and sensitive molecular diagnostic system.