| Autor: |
Lesinski JM; Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland., Khosla NK; Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland., Paganini C; Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland., Verberckmoes B; Faculty of Medicine and Health Sciences, Department of Public Health and Primary Care, Ghent University, De Pintelaan 185, 9000 Gent, Belgium., Vermandere H; Faculty of Medicine and Health Sciences, Department of Public Health and Primary Care, Ghent University, De Pintelaan 185, 9000 Gent, Belgium., deMello AJ; Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland., Richards DA; Institute for Chemical and Bioengineering, ETH Zurich, Vladimir-Prelog-Weg 1, 8093 Zürich, Switzerland. |
| Abstrakt: |
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. |
