Zwerus, Jordy T.Berghuis, Nicole F.Jacques, Jeroen M.Mars-Groenendijk, RoosBusker, Ruud W.Paauw, Armandde Jong, Ad L.van Leeuwen, Hans C.2024-09-122024-09-122024Zwerus JT, Berghuis NF, Jacques JM, Mars-Groenendijk R, Busker RW, Paauw A, et al. A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels. Biosens Bioelectron. 2024 Oct 1;261:116464. DOI: 10.1016/j.bios.2024.1164640956-5663http://hdl.handle.net/10230/61064Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring.application/pdfeng© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).info:eu-repo/semantics/articlehttp://dx.doi.org/10.1016/j.bios.2024.116464CRISPR-CasDNA pre-amplificationFRET reporterFluorescent-based assaysTerminal deoxynucleotidyl transferaseinfo:eu-repo/semantics/openAccess