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 the current 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 that are easily visible to the naked eye under UV-light; we named this detection assay CANTRIP: Cas12a Activated Nuclease poly-T Reporter Illuminating Particles. 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’-OH 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 copper nanoparticles (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 copper-nanoparticles into a single reaction, appears to be a robust, low-cost and simple diagnostic tool.
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