FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result.

Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated Protein (CRISPR-Cas) systems have evolved several mechanisms to specifically target foreign DNA. These properties have made them attractive as biosensors. The primary drawback associated with contemporary CRISPR-Cas biosensors is their weak signaling capacity, which is typically compensated for by coupling the CRISPR-Cas systems to nucleic acid amplification. An alternative strategy to improve signaling capacity is to engineer the reporter, i.e., design new signal-generating substrates for Cas proteins. Unfortunately, due to their reliance on custom synthesis, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we investigate a substrate based on a fluorescein (FAM)-tetramethylrhodamine (TAMRA) Förster resonant energy-transfer (FRET) pair that functions as a seamless "drop-in" replacement for existing reporters, without the need to change any other aspect of a CRISPR-Cas12a-based assay. The reporter is readily available and employs FRET to produce two signals upon cleavage by Cas12a. The use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays when compared to a traditional FAM-Black Hole Quencher (BHQ) quench-based reporter. We comprehensively characterize this reporter to better understand the reasons for the improved signaling capacity and benchmark it against the current standard CRISPR-Cas reporter. Finally, to showcase the real-world utility of the reporter, we employ it in a Recombinase Polymerase Amplification (RPA)-CRISPR-Cas12a DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.

Simplifying the complex: accessible microfluidic solutions for contemporary processes within in vitro diagnostics.

In vitro diagnostics (IVDs) form the cornerstone of modern medicine. They are routinely employed throughout the entire treatment pathway, from initial diagnosis through to prognosis, treatment planning, and post-treatment surveillance. Given the proven links between high quality diagnostic testing and overall health, ensuring broad access to IVDs has long been a focus of both researchers and medical professionals. Unfortunately, the current diagnostic paradigm relies heavily on centralized laboratories, complex and expensive equipment, and highly trained personnel. It is commonly assumed that this level of complexity is required to achieve the performance necessary for sensitive and specific disease diagnosis, and that making something affordable and accessible entails significant compromises in test performance. However, recent work in the field of microfluidics is challenging this notion. By exploiting the unique features of microfluidic systems, researchers have been able to create progressively simple devices that can perform increasingly complex diagnostic assays. This review details how microfluidic technologies are disrupting the status quo, and facilitating the development of simple, affordable, and accessible integrated IVDs. Importantly, we discuss the advantages and limitations of various approaches, and highlight the remaining challenges within the field.