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

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.

Combining CRISPR-Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips.

CRISPR-Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the next-generation diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters. In a previous study, we reported coupling CC-cleavage activity with the enzyme terminal deoxynucleotidyl transferase (TdT) that elongates cleaved ssDNA reporter fragments with dTTP nucleotides. These elongated poly(thymine) tails then act as scaffolds for the formation of copper nanoparticles which generate a bright fluorescent signal upon UV excitation. In the current study, we visualize the poly(thymine) tails on lateral flow strips, using different combinations of biotinylated or fluorescein-labeled nucleotides, various reporters, and capture oligos. One particular approach, using a fluorescein reporter, reached a target sensitivity of <1 pM and was named Cas activity assay on a strip and was tested using Bacillus anthracis genomic DNA.

Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles.

Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP). Upon specific target DNA recognition by Cas12a, single-stranded DNA (ssDNA) reporter oligos with blocked 3'-ends are cut into smaller ssDNA fragments, thereby generating neo 3'-hydroxyl moieties. TdT subsequently elongates these newly formed ssDNA fragments, incorporating only dTTP nucleotides, and these poly(thymine)-tails subsequently function as scaffolds for the formation of CuNPs. These CuNPs produce a bright fluorescent signal upon UV excitation, and thus, this bright orange signal indicates the presence of target DNA, which in this proof-of-concept study consisted of anthrax lethal factor plasmid DNA. CANTRIP, which combines two detection platforms consisting of CRISPR-Cas12a and fluorescent CuNPs into a single reaction, appears to be a robust, low-cost and simple diagnostic tool.