The demand for clinical analyses in hospital is growing, and proteins and nucleic acids (NAs) are the two main biomarker categories that are more relevant for diagnostics . The gold standard techniques for quantitative analysis of the abovementioned biomarkers are the enzyme-linked immunosorbent assay (ELISA) for proteins, the polymerase chain reaction (PCR) for deoxyribonucleic acids (DNA) and reverse transcription PCR (RT-PCR) for ribonucleic acids (RNA). All those techniques require expensive, bulky, and complicated instrumentation to use, making them inaccessible to the average end user at home. The necessity for new point-of-care (POC) devices, over the pregnancy tests, started to become more evident after the COVID pandemic where test kits based on lateral flow enabled to conduct mass testing on the world population: a simple sensing platform provides a positive or negative result without the use of sophisticated instrumentation but just by visual identification. Unfortunately, despite significant effort over the past years, a quantitative POC platform for protein or NAs analysis is still lacking.
In this work, we propose a simple and quantitative protein detection method based on the use of a DNA circuit [1, 2] for the recognition of monoclonal antibodies and the activation of a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) system (Figure 1). The versatility of CRISPR systems, as well as their lower cost, along with their excellent sensitivity and repeatability, make the developed method suitable for a POC application.
The DNA circuit includes an inactive DNA sequence called translator, which contains the Target Sequence (TS) and the complementary single-stranded Protospacer Adjacent Motif (PAM) in a closed loop conformation to prevent the access to the Cas. In addition to the translator, the reaction solution contains two additional single-stranded DNA (ssDNA) oligos called splits: the split 1 which presents the PAM sequence and the TS’s complementary region, and the split 2 which has a toehold region to accelerate the circuit’s activation. The antibody sensing element is a Peptide Nucleic Acid (PNA) which contains the epitope of the monoclonal antibody, hybridized to an extremity of each split. After the antibody-antigen peptides binding, the peptide-DNA splits are colocalized and initiate the strand displacement on the translator. This results in the translator activation in which the PAM is now in a double-stranded conformation. The Cas12a and the CRISPR-RNA (crRNA) ribonucleoprotein (RNP) recognizes the PAM and initiate the specific TS cleavage.
We exploit the Cas12a’s collateral cleavage (trans-cleavage) activity toward single-stranded DNAs to generate a fluorescent signal from a molecular beacon reporter, which is functionalized with a fluorophore and a quencher in our case. Despite the tremendous potential, the system still needs a prior annealing step at different temperatures to improve the repeatability. Thus, our research focuses on the development of a “one-pot CRISPR-based assay” by maintaining the same performances of a multistep one.
The one-pot CRISPR-based assay has the potential to impact modern diagnostics and revolutionize the clinical market by introducing a new tool for NA and protein diagnostics at the POC. We set a first step in this direction by simplifying the assay procedure for future implementation in a POC device for protein detection.
Figure 1: An engineered DNA circuit for the detection of a monoclonal antibody and the activation of the ribonucleoprotein (RNP) made of the Cas12a and the CRISPR-RNA (crRNA) for the recognition of the target sequence (TS). The antibody binding event causes the DNA splits strands-displacement by co-localization. The DNA translator starts to hybridize with the two splits and forms a double-stranded protospacer adjacent motif (PAM), successively the RNP starts the cleavage of the TS. At the same time, other small single-stranded DNA (ssDNA) sequences can diffuse to the accessible active site, and by a collateral cleavage (trans-cleavage) the Cas12a starts to cleave them. We used a molecular beacon ssDNA reporter, fluorophore and quencher terminal labelled, to be able to generate a fluorescence signal after the trans-cleavage.
Quiz question
Is the DNA circuit activating spontaneously?
Yes, slowly (>1 day). The antibody reduces the reaction time.
Yes, quickly (minutes). The antibody reduces the reaction time.
No, only the antibody can trigger the DNA circuit activation.