One-Pot Diagnostic Methods Based on CRISPR/Cas and Argonaute Nucleases

Life Science

Life science

1. Microfluidic Chip Technology

Physical isolation is the most direct method to achieve “one-pot” CRISPR detection, commonly achieved by using special isolation media to fix amplification and cleavage reactions in two chambers, with manual operation to mix the cleavage system and amplification products after amplification. However, this approach still faces challenges such as reagent fixation stability and operational complexity. Microfluidic chips integrate multiple reaction chambers into a single reaction carrier, driven by external forces to achieve physical separation of amplification and nuclease reactions, enhancing sensitivity, multiplexing, and automation levels. For example, the DISCoVER system, which utilizes gravity to drive amplification products into the Cas reaction system, has achieved high sensitivity and multiplex detection of SARS-CoV-2. Pressure-driven microfluidic biosensors (FA-MB) and centrifugal microfluidic chips provide efficient fluid handling and new methods for multiplex “one-pot” detection. Although the development of microfluidic chips is complex and costly, advancements in 3D printing and reduced material costs indicate that there is still significant room for improvement in this method.

2. Component Optimization

Optimizing the components of the reaction system is also an important way to achieve “one-pot” CRISPR detection. By adjusting the concentrations of primers, crRNA, Cas proteins, dNTPs, metal ions, or adding additional auxiliary factors such as RNase H and RNase inhibitors in the reaction buffer, the balance between amplification and Cas cleavage efficiency can be regulated, improving sensitivity and detection efficiency. For example, STOPCovid V2 optimized the required components of the Cas cleavage system and systematically screened buffer components to achieve high sensitivity detection of SARS-CoV-2. Additionally, choosing amplification systems that are more compatible with Cas protein reaction conditions, such as transcription-mediated amplification (TMA) and rolling circle amplification (RCA), can also enable effective “one-pot” detection. Standardized and streamlined experimental design (DOE) can maximize optimization efficiency. Although component optimization does not require specialized consumables and instrument design, the optimization process remains complex due to the varying efficiencies of amplification and cleavage for different targets, and the generalizability of the optimized system still requires further validation with a larger number of targets.

3. Mining and Engineering of Cas Proteins

Mining and engineering of Cas proteins can yield novel Cas proteins with higher tolerance to amplification systems, particularly enhancing their thermal stability, which is an important direction for improving Cas proteins. Novel thermally stable Cas proteins (such as AacCas12b and BrCas12b variants) can withstand higher temperatures, making them compatible with the reaction conditions of LAMP amplification technology. Additionally, constructing protein mutants using artificial intelligence and machine learning can enhance thermal stability and reduce non-specific template DNA binding capacity, improving sensitivity and robustness of detection. With the continuous elucidation of the structure-function relationship of proteins and the development of high-throughput protein screening technologies, directed evolution to modify the activity of Cas proteins may become an important avenue for improving the efficiency of “one-pot” detection.

4. Modification of Guide RNAs

Modifying guide crRNA to regulate Cas12a activity is another method to achieve “one-pot” detection. Light-controlled blocking nucleic acids, suboptimal PAM, and no PAM designs help to address the problem of nucleic acids being prematurely cleaved by Cas12a, reducing the interference of Cas proteins on primers and templates in the amplification system, thereby improving overall efficiency and sensitivity.

5. Ago-Based “One-Pot” Detection Technology

Compared to CRISPR technology, Ago-based detection technology is still in its early stages, but it has shown significant application potential due to its characteristics of short ssDNA guide strands and single-enzyme target detection. Utilizing the unique properties of thermophilic Ago proteins, combined with custom consumables for physical isolation, the SPOT system has achieved temperature-controlled “one-pot” detection, while another OPTIMAL system has developed heat-activated immobilized Ago enzymes to enable stepwise initiation of amplification and cleavage reactions within conventional reaction tubes. These innovations provide economical, efficient, and robust solutions for nucleic acid detection and are expected to expand into more POCT application scenarios.

Summary and Outlook

“One-pot” detection technologies based on CRISPR and Ago demonstrate broad application prospects in infectious disease diagnosis, environmental monitoring, and agriculture. Despite their immense potential, challenges such as specialized equipment consumables, optimization complexity, and generalizability validation still need to be addressed. Future research will focus on the discovery of novel enzymes, enhancement of enzyme formulation controllability, and miniaturization and automation of supporting equipment. The development of artificial intelligence, protein engineering, and electrochemical biosensors will further improve development cycles, enzymatic performance, and usability, driving the evolution of “one-pot” detection technologies and developing more efficient, sensitive, and user-friendly next-generation molecular diagnostic tools in the field of point-of-care testing.

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