Coupling nucleic acid circuitry with the CRISPR-Cas12a system for universal and signal-on detection

Abstract

We report a universal and signal-on HCR based detection platform via innovatively coupling the CRISPR-Cas12a system with HCR. By using this CRISPR-HCR pathway, we can detect different targets by only changing the crRNA. The CRISPR-HCR platform coupling with an upstream amplifier can achieve a practical sensitivity as low as ∼aM of ASFV gene in serum.

Scheme 1. Schematic illustration of the CRISPR-HCR platform. (a) Principle of CRISPR-Cas12a-mediated cis and trans cleavage of DNA. (b and c) CRISPR-HCR platform. Without the target sequence, I₀ activates the HCR assembly and produces low fluorescence background due to FRET (b). After target binds with crRNA, Cas12a cleaves I₀ and remains HCR unreacted, producing increased fluorescence targeting signal (c).

Fig. 1. Performance verification for CRISPR-HCR platform. (a) Signal (fluorescence spectra) comparison between traditional HCR and CRISPR-HCR strategy. (b) 3D-Barograph for the optimization of the signal-to-background ratio of CRISPR-HCR. (c) Barograph of the fluorescence intensity to validate the universality of the CRISPR-HCR with different targets, including dsDNA-T1 (blue), dsDNA-T2 (orange) and ssDNA-T (purple), the real-time fluorescence signal has been shown in Fig. S2. The error bars represent standard deviation from two independent tests.

Fig. 2. Coupling LAMP with CRISPR-HCR platform (LAMP-CRISPR-HCR). (a) Schematic illustration of CRISPR-HCR system coupling with LAMP reaction. (b) Fluorescence spectra of CRISPR-HCR for ASFV-LAMP-products amplified from different amounts of synthetic dsDNA-ASFV. (c) Fluorescence spectra of the CRISPR-HCR for LAMP products amplified from specific target (5000 copies per μL dsDNA-ASFV), and non-specific targets (water control “NC”, 20 000 copies per μL synthetic dsDNA-MERS and dsDNA-M13mp18). dsDNA-MERS and dsDNA-M13mp18 is, in respective, the synthetic DNA sequence segmented from MERS-CoV and M13mp18 gene. (d) Fluorescence spectra of CRISPR-HCR for ASFV-LAMP-products amplified from water control “NC” and different dilutions of the dsDNA-ASFV extracts in pig serum sample.

Fig. 3. Verification of the flexibility via coupling CRISPR with CHA circuit (CRISPR-CHA). (a and b) Schematic illustration of CRISPR-CHA. Without the target sequence, C1 activates the CHA assembly and produces low fluorescence back-ground due to FRET (a). After target binds with crRNA, Cas12a cleaves C1 and remains CHA unreacted, producing increased fluorescence targeting signal (b). (c) Fluorescence kinetic curves of CRISPR-CHA without (“NC”) and with different concentrations of dsDNA-T1, in presence of C1, H3, H4-FAM and BlackQ1-P:cP. (d) Barograph of the fluorescence intensity of CRISPR-CHA detection for the ASFV-LAMP-products amplified from water control (“LAMP-CRSIPR-CHA NC”) and 500 copies per μL dsDNA-ASFV (“LAMP-CRSIPR-CHA PC”). The error bars represent standard deviation from two independent tests.