Recent Advances in CRISPR/Cas-Based Biosensors for Protein Detection

Abstract

CRISPR is an acquired immune system found in prokaryotes that can accurately recognize and cleave foreign nucleic acids, and has been widely explored for gene editing and biosensing. In the past, CRISPR/Cas-based biosensors were mainly applied to detect nucleic acids in the field of biosensing, and their applications for the detection of other types of analytes were usually overlooked such as small molecules and disease-related proteins. The recent work shows that CRISPR/Cas biosensors not only provide a new tool for protein analysis, but also improve the sensitivity and specificity of protein detections. However, it lacks the latest review to summarize CRISPR/Cas-based biosensors for protein detection and elucidate their mechanisms of action, hindering the development of superior biosensors for proteins. In this review, we summarized CRISPR/Cas-based biosensors for protein detection based on their mechanism of action in three aspects: antibody-assisted CRISPR/Cas-based protein detection, aptamer-assisted CRISPR/Cas-based protein detection, and miscellaneous CRISPR/Cas-based methods for protein detection, respectively. Moreover, the prospects and challenges for CRISPR/Cas-based biosensors for protein detection are also discussed.

Keywords: CRISPR; biosensors; detection; protein.

Figure 2

The strategies for the aptamer-assisted CRISPR/Cas-based detection of protein with nucleic acid amplification. (a) A schematic diagram of the nicking enzyme-free SDA-assisted CRISPR/Cas colorimetric detection of PSA. Reproduced with permission from [83]. Copyright 2022 Elsevier. (b) A schematic illustration of the aptamer-CRISPR/Cas12a assay with PCR amplification. Reproduced with permission from [87]. Copyright 2021 Elsevier. (c) A schematic diagram of the detection of TEV surface proteins by apta-HCR-CRISPR. Reproduced with permission from [89]. Copyright 2022 Elsevier.

Figure 3

The strategies for aptamer-assisted CRISPR/Cas biosensors for protein detection without amplification. (a) A schematic diagram of the dual aptamer-based CRISPR/Cas12a biosensor for synergistic sensing of SARS-CoV-2 antigen detection without PCR amplification. Reproduced with permission from [92]. Copyright 2021 Elsevier. (b) A schematic diagram of the DNA walker amplified “one-to-many” CRISPR/Cas12a-mediated fluorescent biosensor for detecting CEA. Reproduced with permission from [93]. Copyright 2022 Elsevier. (c) A schematic diagram of a multifunctional biosensing platform combining CRISPR/Cas12a and the aptamer for detecting AFP. Reproduced with permission from [94]. Copyright 2021 Elsevier. (d) A schematic illustration of the regulation of the trans-cleavage activity of CRISPR/Cas13a by the ssDNA blocker at low Mg²⁺ concentration for protein detection. Reproduced with permission from [95]. Copyright 2022 American Chemical Society. (e) A schematic illustration of E-CRISPR for protein detection. Reproduced with permission from [96]. Copyright 2019 John Wiley and Sons. (f) A schematic illustration of Nano-CLISA for protein detection. Reproduced with permission from [112]. Copyright 2021 Elsevier.

Figure 4

The strategies for the miscellaneous CRISPR/Cas-based methods for protein detection. (a) A schematic illustration of pcDNA-based Cas12a for detecting telomerase activity in living cells. Reproduced with permission from [117]. Copyright 2021 Royal Society of Chemistry. (b) A schematic diagram of ExoIII-protected CRISPR/Cas12a-based biosensor for detecting TFs in cancer cells. Reproduced with permission from [120]. Copyright 2021 Elsevier. (c) A schematic diagram of the PR-Cas detection of proteases. Reproduced with permission from [121]. Copyright 2020 Springer Nature. (d) A schematic diagram of a strategy to recognize protein/small molecule interactions based on CRISPR/Cas12a trans cleavage activity. Reproduced with permission from [122]. Copyright 2021 Elsevier.

Scheme 1

A schematic illustration of CRISPR/Cas-based biosensors for protein detection.

Figure 1

The strategies for antibody-based CRISPR biosensors for protein detection. (a) Schematic illustration of the CRISPR/Cas13a signal amplification linked immunosorbent assay (CLISA). Reproduced with permission from [61]. Copyright 2020 American Chemical Society. (b) Schematic illustration of the detection of antibodies using proximity CRISPR/Cas12a integrated with nicking cleavage. Reproduced with permission from [63]. Copyright 2021 Royal Society of Chemistry.