ABSTRACT
The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTS: Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches.Understanding Cas13a's allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants.These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains.
ABSTRACT
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.
