FRETting about CRISPR-Cas assays: Dual-channel reporting lowers detection limits and times-to-result

21 June 2024, Version 2
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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. This adds time and complexity to the diagnostic process, limiting the practicality of these assays for many clinical applications. 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 or specialized analytical equipment, most of these engineered reporter substrates are inaccessible to many researchers. Herein, we present a substrate for Cas12a 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 a FRET pair to produce two signals upon cleavage by Cas12a. Use of both signals in a ratiometric manner provides for improved assay performance and a decreased time-to-result for several CRISPR-Cas12a assays. 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 our reporter, we apply it to the analysis of Human papillomavirus in clinical samples

Keywords

CRISPR
CRISPR-Cas
Cas12a
Point-of-care diagnostics
FRET
Diagnostic
HPV-16
Biosensors
Molecular diagnostics

Supplementary materials

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Experimental methods and additional figures
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