Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters

Abstract

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Graphical Abstract

Here we report on the molecular basis for the enhancement of trans-cleavage activity of Cas12a enzyme using hairpin DNA as FRET reporters.

Figure 1.

Schematic for Cas12a trans-cleavage activity based on hairpin DNA reporters. We explore the use of different FRET-based hairpin DNA reporters as signal transducers of the Cas12a-based trans-cleavage reaction. The hairpin structure separates the single-stranded portion that preferentially can be cleaved by activated Cas12a proteins (the loop) from those that define the switching equilibrium and signal transduction (the stem). Once the ternary complex of Cas12a/crRNA/dsDNA is formed, the trans-cleavage activity is activated and the hairpin DNA reporter can be cleaved, thus generating a fluorescence signal change.

Figure 2.

Design and characterization of different hairpin DNA reporters as FRET-based reporters of LbCas12a trans-cleavage activity. (A) We tested three different hairpin DNA reporters having same stem composition (3GC + 2AT) but different loop length and an increasing number of thymine (from 5 to 30). All the DNA reporters were functionalized with 6-FAM at 5′-end and with BHQ-1 at 3′-end. Melting curves indicate that by increasing the number of thymine in the loop we can rationally control the melting temperatures of the reporters and thus the stability of the hairpin structure. (B) LbCas12a trans nuclease activity on linear ssDNA and hairpin DNA reporters. The fluorescence kinetic analysis over time of LbCas12a trans-cleavage activity triggered by a fully complementary 20bp-long double stranded DNA activator show enhanced signal transduction using the Stem-loop #10T reporter. (C) Fluorescence fold change as a function of the temperature after 1h of LbCas12a trans-cleavage activity. (D) Effect of Mg²⁺ concentration on LbCas12a-based trans-cleavage activity. The assays were performed by adding 4.5 μl of LbCas12a/crRNA complex (200 nM) preassembled with the dsDNA activator (10 nM) to a solution containing the DNA reporter (100 nM). The trans-cleavage reactions in c) and d) were stopped after 1 h by heating the solutions at 65°C for 10 min. All bar graph shows the mean fold change calculated as F – F₀/F₀ (background-subtracted fluorescence divided by background fluorescence of the DNA reporter at time t = 0) ± SD, where n = 3 replicates.

Figure 3.

Kinetic analysis of LbCas12a trans-cleavage activity. (A) Time-dependent trans-cleavage activity of activated LbCas12a on FRET-labelled DNA reporters (250 nM). Substrate (e.g. DNA reporters, S) and degraded products (P) were resolved by polyacrylamide gel electrophoresis (PAGE). The intrinsic fluorescence background of the DNA reporters (lines 1) and signal change over time is consistent with the increased amount of degraded oligonucleotides over time (lines 3 to 8). (B) Fraction of DNA reporters cleaved by the active LbCas12a/crRNA/dsDNA complex (RNP complex, final concentration 20 nM/20 nM/1 nM) vs time. Fluorescence assays were performed at 37°C by adding 4.5 μl of a 10x concentrated RNP complex to a solution containing the DNA reporter (final concentration 100 nM). (C) Representative Michaelis-Menten plots for LbCas12a trans-cleavage activity on the different linear/hairpin DNA reporters using a dsDNA activator. Measured K_m, k_cat and k_cat/K_m values reported as mean ± SD, where n = 3.

Figure 4.

Mechanistic insights from molecular simulations. (A) Long timescale molecular dynamics (MD) simulations reporting a large-scale opening of the Nuc (indicated by the arrow in green). (B) Outward bending of Nuc along ∼4 μs MD trajectories (in two replicates), shown through the time evolution of the center of mass distance between the REC2 and Nuc domains (top), and the angle of Nuc with respect to the RuvC domain (bottom). (C) Comparison of the binding free energy between the association of the stem loop (green) and linear ssDNA (purple) with Cas12a using the MM-GBSA solvation method.

Figure 5.

Sensitivity and specificity assays. (A) Amplification free, direct detection of SARS-CoV-2 ds-DNA amplicons. Calibration curves obtained after 1h of incubation of different concentrations of ds-DNA amplicons to a solution containing LbCas12a/crRNA (20 nM, final concentration) and the Linear-ssDNA or the Stem-loop #10T DNA reporter (100 nM). All the values reported show the mean ± SD, where n = 3 replicates. (B) Two-pot CRISPR-based detection of PCR amplified SARS-CoV-2 ds-DNA amplicons at known concentrations showing enhanced signal transduction using the Stem-loop #10T reporter. Pairwise comparisons of reactions containing target ds-DNA amplicons with the control (0 copies per μl of ds-DNA amplicon) were done using an unpaired t-test with Welch's correction (n = 5). The asterisks denote values levels of statistical significance: P< 0.0001 (****); P< 0.001 (***); P< 0.001 (**); p ≤ 0.005 (*). (C) Detection of PCR-amplified Salmonella Typhimurium samples, realized by adding diluted PCR-amplified Salmonella Typhimurium samples to a solution containing LbCas12a/crRNA (20 nM, final concentration) and the linear-ssDNA or the Stem-loop #10T DNA reporter (500 nM). Kinetic assays show enhanced signal transduction for stem-loop #30T reporter testing samples at different dilution factors (1:10^² and 1:10^⁴). (D) Fold change of two-pot CRISPR-based detection of PCR-amplified Salmonella Typhimurium at different dilution factors. Pairwise comparisons of reactions containing target PCR-amplified Salmonella amplicons with the control (0 copies per μl of DNA amplicon) were done using an unpaired t-test with Welch's correction (n = 5). The asterisks denote values levels of statistical significance: P< 0.0001 (****); P< 0.001 (***); P< 0.001 (**); P ≤ 0.005 (*). Comparisons that were not significant (NS) have a P value of >0.05 or signal that was lower than the control (0 copies per μl of DNA amplicon). (E) Specificity test on single-nucleotide mismatch mutations (from MM1 to MM20) and double-nucleotide mismatch mutations (from MM1-2 to MM19-20) on synthetic SARS-CoV-2 dsDNA activator. Kinetic assays report the fluorescence difference between perfect match (PM) and mismatch 1 (MM1) target (right, t = 4 min). (F) Superimposed bar graphs showing the difference between the fold change related to the perfect match (PM) and to the mismatch (MM) target (right, 10 nM at t = 4 min). All the values reported show the mean ± SD, where n = 3 replicates.