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

Abstract

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.

Keywords: COPPER nanoparticles; CRISPR CAS; DNA detection.

Figure 1:

schematic representation of the CANTRIP DNA detection assay. See text for further details.

Figure 2:

CuNP fluorescence emission intensity of the CANTRIP assay on anthrax lethal factor template gene recognition. To verify the correct coupling of the Cas12a target activation via poly-T scaffolded CuNPs formation, a synthetic ALF gene was detected. Three different crRNA targeting ALF gene were compared, and the negative controls consisted of leaving out individual components of the assay and a non-targeting crRNA (nt crRNA). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. RepB = blocked reporter. Inset: Fluorescence emission image upon illumination with UV light corresponding to bar chart (excluding nt crRNA).

Figure 3:

sensitivity of the fluorescent CANTRIP assay using serial dilution of anthrax lethal factor plasmid as the target dsDNA in combination with crRNA2. The negative control contained no ALF target plasmid (0 pM). Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. Inset: Corresponding fluorescence emission image upon illumination with UV light. Image was rearranged to match the bar graph.

Figure 4:

variation of incubation times for the complete CANTRIP assay reaction using crRNA2. Incubation time with the Cas12a(Cas) enzyme was 15, 30 or 60 min. Incubation time with TdT was 1, 2 or 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments. Inset: Corresponding fluorescence emission image upon illumination with UV light.

Figure 5:

CANTRIP can be performed in a two-step reaction without Cas12a heat inactivation. Pre-incubation time with the Cas12a enzyme, before TdT addition, was 60 or 0 min after which the Cas12a reaction mix was heat inactivated at 70°C or not. Subsequent incubation time with TdT was 3 h. Bars represent the mean of two technical duplicates of an experiment and the corresponding differences between these two experiments.