FRETting about CRISPR-Cas Assays: Dual-Channel Reporting Lowers Detection Limits and Times-to-Result
Jake M. Lesinski, Nathan Khosla, Carolina Paganini, Bo Verberckmoes, Heleen Vermandere, Andrew J. deMello, Daniel A. Richards
Abstract
High Resolution Image Download MS PowerPoint Slide C lustered R egularly I nterspaced S hort P alindromic R epeats- C RISPR- A ssociated P rotein (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 R ecombinase P olymerase A mplification (RPA)–CRISPR-Cas12a D NA E ndonuclease- T arget e d C RISPR T rans R eporter (DETECTR) assay to detect Human papillomavirus in patient-derived samples.