CRISPR-Cas-based techniques for pathogen detection: Retrospect, recent advances, and future perspectives

Abstract

Background: Early detection of pathogen-associated diseases are critical for effective treatment. Rapid, specific, sensitive, and cost-effective diagnostic technologies continue to be challenging to develop. The current gold standard for pathogen detection, polymerase chain reaction technology, has limitations such as long operational cycles, high cost, and high technician and instrumentation requirements.

Aim of review: This review examines and highlights the technical advancements of CRISPR-Cas in pathogen detection and provides an outlook for future development, multi-application scenarios, and clinical translation.

Key scientific concepts of review: Approaches enabling clinical detection of pathogen nucleic acids that are highly sensitive, specific, cheap, and portable are necessary. CRISPR-Cas9 specificity in targeting nucleic acids and "collateral cleavage" activity of CRISPR-Cas12/Cas13/Cas14 show significant promise in nucleic acid detection technology. These methods have a high specificity, versatility, and rapid detection cycle. In this paper, CRISPR-Cas-based detection methods are discussed in depth. Although CRISPR-Cas-mediated pathogen diagnostic solutions face challenges, their powerful capabilities will pave the way for ideal diagnostic tools.

Keywords: CRISPR-Cas; Pathogen detection; Point-of-care testing; Standardized testing.

Graphical abstract

Fig. 1

Combining nucleic acid amplification and the CRISPR-Cas reaction in a “one-pot” detection method: A. nucleic acid amplification technique and the Cas reaction are integrated on the device. B. Cas enzyme is linked to the tube's wall and centrifuged into the nucleic acid amplification system.

Fig. 2

Detection methods combined CRISPR-Cas with biosensors: A. Method of combining dCas9 with nanopores; B. Method of combining Cas13 with microfluidic chip; C. Method of combining Cas13 with allosteric probe.

Fig. 3

A. Mulitplexing detection design: Using different Cas proteins which have different cutting preferences for reporters to achieve multiplexing detection. B.Use Ni-NTA functionalized glass surface and SG I to realize a fluorescent reporter system; C. Use UiO66 to quench/recover the fluorescent reporter under different conditions to realize a fluorescent reporter system; D. The workflow of CARMEN-Cas13: Variety of fluorescent colors provides the basis for multiplexing of CRISPR-Cas-related nucleic acid detection technology.

Fig. 4

Non-fluorescence-based reporting method: A. Lateral flow reporting system of CIA platform: The structure and results display of lateral flow reporting system on CIA; B. The combination of Cas9 and toe switch can cause the colorimetric system to change through chemical reaction to realize the result output; C. The combination of SMR sensor and dCas9-sgRNA-target DNA causes wavelength shift to achieve result output.

Fig. 5

Summary of CRISPR-based nucleic acid detection methods for pathogens.