Harnessing CRISPR/Cas Systems for DNA and RNA Detection: Principles, Techniques, and Challenges

Abstract

The emergence of CRISPR/Cas systems has revolutionized the field of molecular diagnostics with their high specificity and sensitivity. This review provides a comprehensive overview of the principles and recent advancements in harnessing CRISPR/Cas systems for detecting DNA and RNA. Beginning with an exploration of the molecular mechanisms of key Cas proteins underpinning CRISPR/Cas systems, the review navigates the detection of both pathogenic and non-pathogenic nucleic acids, emphasizing the pivotal role of CRISPR in identifying diverse genetic materials. The discussion extends to the integration of CRISPR/Cas systems with various signal-readout techniques, including fluorescence, electrochemical, and colorimetric, as well as imaging and biosensing methods, highlighting their advantages and limitations in practical applications. Furthermore, a critical analysis of challenges in the field, such as target amplification, multiplexing, and quantitative detection, underscores areas requiring further refinement. Finally, the review concludes with insights into the future directions of CRISPR-based nucleic acid detection, emphasizing the potential of these systems to continue driving innovation in diagnostics, with broad implications for research, clinical practice, and biotechnology.

Keywords: CRISPR/Cas; biosensing; diagnosis; nucleic acids detection.

Figure 4

Schematics for CRISPR-based detection systems for pathogenic and non-pathogenic DNA. The detection systems are categorized by signal readout methods. HOLMES, CDetection, E-CRISPR, CASLFA, GPHOXE, DNA-FISH, and metal-enhanced fluorescence detection are illustrated [23,27,29,34,37,88,89].

Figure 5

Schematics for CRISPR-based detection systems for pathogenic RNA. The detection systems are categorized by signal readout methods. SHERLOCK, DETECTR, FELUDA, CARMEN, and SARS-CoV-2 detection platforms using Cas12a and Cas13a are illustrated [41,43,46,49,61,77,78].

Figure 6

Schematics for CRISPR-based detection systems for non-pathogenic RNA. The detection systems are categorized by signal readout methods. RACE, MICR-ON, PECL-CRISPR, COMET, EXTRA-CRISPR, and RCH are illustrated [50,51,52,53,56,87].

Figure 1

Molecular mechanism of CRISPR-associated nucleic acid detection. The Cas protein-gRNA complex first forms and binds to the target nucleic acid. Different Cas proteins bind depending on the type of target. The ribonucleoprotein (RNP) complex then cleaves the target, a process known as cis-cleavage. While the cleavage reaction mediated by Cas9 terminates at this stage, other Cas proteins, including Cas12, Cas13, and Cas14, further exhibit nonspecific cleavage of nearby nucleic acids, referred to as trans-cleavage. This trans-cleavage activity is a key mechanism for signal amplification in CRISPR-based diagnostics.

Figure 2

Signal production systems used in CRISPR-based nucleic acid detection. The fluorescent reporter system relies on energy transfer between a fluorophore (F) and a quencher (Q), where fluorescence is generated upon cleavage of the reporter. The electrochemical reporter system measures changes in electrical signals dependent on the distance between a redox molecule and gold electrode. Intercalating dyes, such as SYBR Green, bind to double-stranded DNA and emit fluorescence upon intercalation. Gold nanoparticles serve as versatile reporters; they can be visualized by color changes through aggregation visible to the naked eye and also be conjugated with a variety of molecules, including antibodies, proteins, nucleic acids, fluorophores, and chemicals, for diverse biosensing applications.

Figure 3

Signal readout methods used in CRISPR-based nucleic acid detection. Three major readout methods are illustrated: fluorescence, electrochemical, and colorimetric. In fluorescence readouts, the Cas-gRNA complex cleaves fluorescent reporters, producing a fluorescence signal. The graph shows the increase in fluorescence over time for positive sample (POS) compared to negative samples (NEG). In electrochemical readouts, redox molecules are conjugated to nucleic acids and immobilized on a gold electrode. When CRISPR complex interacts with the redox-labeled nucleic acids, a detectable change in current is produced, as shown in the voltage-current graph for positive and negative samples. The colorimetric readout, typically in the form of a lateral flow assay (LFA), reports positive results by capturing cleaved products labeled with fluorescein, producing visible bands on the test line, while biotin-labeled parts produce visible bands on the control line.