Self-Supplying Guide RNA-Mediated CRISPR/Cas12a Fluorescence System for Sensitive Detection of T4 PNKP

Abstract

Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.

Keywords: CRISPR/Cas12a; T4 PNKP; transcription.

Figure 1

The principle of the self-supplying guide RNA-mediated CRISPR/Cas12a fluorescence system for sensitive detection of T4 PNKP.

Figure 2

Fluorescence spectra of the proposed T4 PNKP sensor under different conditions: in the presence of T4 PNKP (black curve, B); in the absence of T4 PNKP (red curve, A).

Figure 3

Non-denaturing polyacrylamide gel electrophoresis analysis of the proposed biosensor for T4 PNKP under different conditions. (A) Lane M, DNA Marker; Lane (1) msg; (2) sg; (3) msg +sg; (4) msg +sg +T4PNKP+ λ exo; (5) msg +sg + λ exo; (6) msg +sg +T4PNKP+ λ exo+ T7 RNA polymerase; (7) msg +sg + λ exo+ T7 RNA polymerase; (B) Lane M, DNA Marker; Lane 1-3: (1) msg +sg +T4PNKP+ λ exo+ T7 RNA polymerase+PR30+SS1; (2) msg +sg + λ exo+ T7 RNA polymerase+PR 30+SS1; (3) SS1.

Figure 4

Effect of the (A) phosphorylation reaction time, (B) λ exo concentration, (C) dsDNA concentration, (D) T7 RNA polymerase concentration, (E) PR-30 concentration, and (F) Cas12a concentration on the fluorescence difference. Other reaction conditions: ATP, 10 mM; F-Q Probe, 1 μM. Error bars mean standard deviations (n = 3).

Figure 5

(A) Fluorescence spectra measured by different concentrations of T4 PNKP; (B) the relationship between the linear range of fluorescence intensity and T4 PNKP concentration. Reaction conditions: dsDNA, 1 μM; ATP, 10 mM; λ exo, 5 U/μL; phosphorylation reaction time, 45 min; T7 RNA polymerase, 20 U/μL; Cas12a, 0.46 μM; PR-30, 2 μM; F-Q, 1 μM. Error bars mean standard deviations (n = 3).

Figure 6

The fluorescent intensity induced by T4 PNK and other alternatives: T4 PNKP, T4 PNKP (inactive), UDG, and APE 1. Error bars mean standard deviations (n = 3).