A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels

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• dc.contributor.author Zwerus, Jordy T.
• dc.contributor.author Berghuis, Nicole F.
• dc.contributor.author Jacques, Jeroen M.
• dc.contributor.author Mars-Groenendijk, Roos
• dc.contributor.author Busker, Ruud W.
• dc.contributor.author Paauw, Armand
• dc.contributor.author de Jong, Ad L.
• dc.contributor.author van Leeuwen, Hans C.
• dc.date.accessioned 2024-09-12T06:59:30Z
• dc.date.available 2024-09-12T06:59:30Z
• dc.date.issued 2024
• dc.description.abstract Recent 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.
• dc.format.mimetype application/pdf
• dc.identifier.citation Zwerus 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.2024.116464
• dc.identifier.doi http://dx.doi.org/10.1016/j.bios.2024.116464
• dc.identifier.issn 0956-5663
• dc.identifier.uri http://hdl.handle.net/10230/61064
• dc.language.iso eng
• dc.publisher Elsevier
• dc.relation.ispartof Biosens Bioelectron. 2024 Oct 1;261:116464
• dc.rights © 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/).
• dc.rights.accessRights info:eu-repo/semantics/openAccess
• dc.rights.uri http://creativecommons.org/licenses/by-nc/4.0/
• dc.subject.keyword CRISPR-Cas
• dc.subject.keyword DNA pre-amplification
• dc.subject.keyword FRET reporter
• dc.subject.keyword Fluorescent-based assays
• dc.subject.keyword Terminal deoxynucleotidyl transferase
• dc.title A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels
• dc.type info:eu-repo/semantics/article
• dc.type.version info:eu-repo/semantics/publishedVersion