Novel Anti-CRISPR-Assisted CRISPR Biosensor for Exclusive Detection of Single-Stranded DNA (ssDNA)

Abstract

Nucleic acid analysis plays an important role in disease diagnosis and treatment. The discovery of CRISPR technology has provided novel and versatile approaches to the detection of nucleic acids. However, the most widely used CRISPR-Cas12a detection platforms lack the capability to distinguish single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). To overcome this limitation, we first employed an anti-CRISPR protein (AcrVA1) to develop a novel CRISPR biosensor to detect ssDNA exclusively. In this sensing strategy, AcrVA1 cut CRISPR guide RNA (crRNA) to inhibit the cleavage activity of the CRISPR-Cas12a system. Only ssDNA has the ability to recruit the cleaved crRNA fragment to recover the detection ability of the CRISPR-Cas12 biosensor, but dsDNA cannot accomplish this. By measuring the recovered cleavage activity of the CRISPR-Cas12a biosensor, our developed AcrVA1-assisted CRISPR biosensor is capable of distinguishing ssDNA from dsDNA, providing a simple and reliable method for the detection of ssDNA. Furthermore, we demonstrated our developed AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of helicase and screen its inhibitors.

Keywords: AcrVA1; CRISPR-based biosensor; Cas12a (cpf1) nuclease; anti-CRISPR proteins; single-stranded DNA (ssDNA).

Figure 1

(a) Schematic illustration of the CRISPR-Cas12a biosensor to detect ssDNA and dsDNA. (b) The relative fluorescence responses of the CRISPR-Cas12a biosensor to detect ssDNA and dsDNA. (c, d) The inhibition effect of AcrVA1, AcrVA4, and AcrVA5 on the performances of the CRISPR-Cas12a biosensor to detect ssDNA and dsDNA, respectively. All experiments were performed with at least three replicates, and the error bars represent the standard deviation. F₁ represents the florescence intensity at the end point, while F₀ represents the initial fluorescence intensity.

Figure 2

(a) Schematic illustration of the AcrVA1-assisted CRISPR-Cas12a biosensor to detect T-ssDNA, NT-ssDNA, and dsDNA. (b–d) Effect of AcrVA1 concentration on the performance of the CRISPR-Cas12a biosensors to detect T-ssDNA, NT-ssDNA, and dsDNA, respectively. (e, f) Detection sensitivity of T-ssDNA using the AcrVA1-assisted CRISPR-Cas12a biosensor. All experiments were performed with at least three replicates, and the error bars represent the standard deviation.

Figure 3

(a) Schematic illustration of the AcrVA1-assisted CRISPR biosensor to monitor the enzymatic activity of UvrD helicase. (b) Relative fluorescence intensity of the AcrVA1-assisted CRISPR biosensors toward the UvrD helicase at concentrations ranging from 0 to 5 μg/mL. (c) Schematic illustration of AcrVA1-assisted CRISPR biosensor to screen the inhibitors of the UvrD helicase. (d) Inhibition efficiency (IE) of different quinone derivatives to the UvrD helicase. (e) Agarose gel electrophoresis to characterize the IE of different quinone derivates of the UvrD helicase. (f) IE toward the concentration of mitoxantrone. All experiments were performed with at least three replicates, and the error bars represent the standard deviation.