Optimizing Dropletized CRISPR-Cas13 Viral Diagnostics using Quantum Nanoparticles

Abstract

CRISPR-Cas13 viral diagnostics leverage the collateral cleavage activity of Cas13, post-activation by the target-specific sequence, to achieve fluorescence detection of an RNA-linked fluorescent reporter (most commonly, the FAM hexauridine probe). Recent innovations in this field have demonstrated that Cas13-based diagnostics can achieve massively multiplexed detection abilities with rapid turnaround times, high throughput, and heightened sensitivity. Building upon CARMEN and mCARMEN technologies specifically, this investigation aimed to further expand the current set of AlexaFluor organic dye-based color codes amenable to dropletized Cas13 detection. We harnessed the unique, tunable optoelectronic properties of highly fluorescent nanoparticles called quantum dots into Cas13 detection assays. Preliminary compatibility testing between quantum dots and Cas13 detection reaction revealed challenges in fluorescence quantification on microplate reader, Biotek Cytation5, and illuminated the need for highly sensitive confocal microscopy in achieving fluorescence detection in emission channels specific to the fluorophores used in the reaction.

Currently under development in our labs and at the Broad Institute of MIT and Harvard is CARMENv2, which aims to dropletize Cas13 detection reactions and ligate biotinylated guide crRNAs and AlexaFluor dyes to Streptavidin-coated beads emulsified into beads. The incorporation of quantum dots into CARMENv2 builds additional dimensions to the color code space. Three forms of color codes were prepared in this investigation: ATTO-655 organic dye, Cadmium Telluride (CdTe) 610, and carbodiimide-linked ATTO-655 with CdTe-610. To build the basis of multiplexing, single-core double emulsion (SCDE) dropletization of Cas13 detection reaction, each with one of these color codes was performed. Subsequent confocal microscopy and imaging analysis of SCDE droplets containing Cas13 detection reaction further revealed that fluorescence signal from both the FAM-hexauridine probe and the droplet-specific color code was successfully detected. The far-reaching impacts of this investigation lie in the pressing demand for point-of-need viral diagnostics that are highly deployable and rapidly scalable to curb infectious disease outbreaks and improve global pandemic preparedness measures.