Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

Abstract

CRISPR/Cas technology originated from the immune mechanism of archaea and bacteria and was awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Molecular diagnostics is highly valued globally for its development as a new generation of diagnostic technology. An increasing number of studies have shown that CRISPR/Cas technology can be integrated with biosensors and bioassays for molecular diagnostics. CRISPR-based detection has attracted much attention as highly specific and sensitive sensors with easily programmable and device-independent capabilities. The nucleic acid-based detection approach is one of the most sensitive and specific diagnostic methods. With further research, it holds promise for detecting other biomarkers such as small molecules and proteins. Therefore, it is worthwhile to explore the prospects of CRISPR technology in biosensing and summarize its application strategies in molecular diagnostics. This review provides a synopsis of CRISPR biosensing strategies and recent advances from nucleic acids to other non-nucleic small molecules or analytes such as proteins and presents the challenges and perspectives of CRISPR biosensors and bioassays.

Keywords: CRISPR/Cas; biosensor; molecular diagnostics; non-nucleic-acid analytes; nucleic acid detection.

FIGURE 1

Examples of three common readout methods. (A) The color of the solution in the centrifuge tube changes due to the aggregation of AuNPs. (B) The reporter consists of a fluorophore and a quencher, linked by a nucleic acid molecule. After its collateral cleavage by the CRISPR system, the change in the distance leads to the generation of fluorescent energy. (C) The conformation of E-DNA changes from hairpin to linear dsDNA when the ssDNA of the gold electrode hairpin structure is complementary to the target ssDNA (red), and Cas12a cleaves the linear DNA to release the MB strand. MB, methylene blue.

FIGURE 2

Strategies for Cas9-based nucleic acid sensor. (A) NASBA amplifies the RNA target, and the trigger sequence for the toehold switch is introduced. Subsequently, the RNA and DNA hybridization produces RNA that is degraded by RNAse H. The second DNA strand is synthesized, and it carries the T7 promoter sequence. The transcribed RNA can be used as starting material for NASBA and interact with the toehold switch. In the presence of PAM sequences, the RNA target site is shorter due to the cleaved template and cannot activate the sensor, and vice versa, producing a significant color change. (B) In the action of Cas9, broken dsDNA at the nicking site is further recognized by the nicking enzyme. Once the DNA polymerase reaches the nicking site, a new strand can be generated and the downstream strand is replaced. As a result, many short ssDNAs are generated to trigger rolling circle amplification (RCA). Subsequently, the RCA product can bind to oligonucleotide-spliced AuNPs, inducing the aggregation of AuNPs. SDA, Strand-displacement amplification. (C) Viral lysate and biotin-PAMmer are added to microplates immobilized with dCas9/gRNA complexes. Following, Streptavidin-HPR and TMB substrate solutions were added to the microplate. Finally, the yellow color is observed in the presence of the virus. (D) The CRISPR-chip is composed of a gFET structure with a complex of sgRNA and dCas9 formed on the graphene surface. When the target DNA is detected, dCas9 binds to the target DNA, which modulates the electrical properties of the gFET and leads to an electrical signal output. gEFT: graphene-based field-effect transistor.

FIGURE 3

Strategies for Cas12 and Cas13-based nucleic acid sensor. (A) After amplification, ssRNA or dsDNA forms dsDNA and produces ssRNA, dsDNA, and ssRNA, depending on the different systems. After being mixed with fluorescent reporters, trans cleavage occurs through specific binding of different CRISPR/Cas systems, which subsequently generates a fluorescent signal. (B) In CONAN, there are three signal processors, namely transducer 1 (T1), transducer 2 (T2), and signal amplifier (A), where the parallel connection of A and T2 forms a positive feedback circuit and T1 is connected in series with the positive feedback circuit. Cas12a and gRNA for target dsDNA (gRNA-T) are pre-assembled as T1, which converts the signal input into active Cas12a protein (Cas12a/gRNA-T/DNA complex). After the action of a self-reporting scgRNA, an amplified fluorescent signal and multiple active (decaged) gRNA molecules are output. T2 contains Cas12a protein and a probe for decaged gRNA, which can transduce the resulting decaged gRNA into another active Cas12a protein to produce an amplified fluorescent signal. scgRNA: switchable-caged guide RNA. (C) The protective oligonucleotide is designed to partially pair with the crRNA, thereby altering the conformation of the crRNA so that it cannot bind to the target DNA and inhibit the cleavage activity of Cas12a. The protective oligonucleotide breaks when exposed to ultraviolet (UV) light at 365 nm isolated from crRNA, thereby restoring the function of CRISPR-Cas12a detection. This system ensures that the target DNA is not reduced by Cas12a during the amplification phase, resulting in a stronger fluorescent signal.

FIGURE 4

Strategies for CRISPR-based non-nucleic-acid detection. (A) Locked fDNA in the presence of a small molecule, one strand binds to it. It releases another complementary DNA strand to participate in the detection reaction (B) The DNAzyme starts in an inactive state and activates its cleavage ability with the involvement of specific ions, thus releasing short strands of DNA to participate in the detection system. (C) Left: Non-nucleic acid as antigen, linked to the CRISPR/Cas system and antibody (yellow)-antigen (green)-antibody (red) structure by a nucleic acid sequence containing the T7 promoter. Right: DNA-AuNP is used instead of antibody and is directly involved in the assay reaction. (D) Immobilized aTF-dsDNA complexes containing CRISPR target sequences change the conformation in the presence of aTF target small molecules, resulting in the release of dsDNA.