The CRISPR system, known for its gene-editing capabilities, has been repurposed into a diagnostic technology. This application uses CRISPR’s components not to alter genes, but to find them, offering a method for detecting specific genetic sequences with high precision. This focus on identifying the presence of DNA or RNA to diagnose conditions is distinct from its therapeutic use. The technology provides a rapid and sensitive platform for identifying diseases.
The Core Mechanism of CRISPR Diagnostics
CRISPR-based diagnostics use two main components: a guide RNA (gRNA) and a CRISPR-associated (Cas) enzyme. The gRNA is engineered to be a mirror image of the target genetic sequence from a source like a virus or a cancerous cell. This guide leads the Cas enzyme directly to the specified DNA or RNA target. While Cas9 is famous for gene editing, diagnostic tools use versions like Cas12 or Cas13, which have properties suited for detection.
Once the guide RNA pairs with its target sequence, the Cas enzyme activates. Instead of making a single, precise cut, the activated enzyme begins to cut other nearby single-stranded nucleic acids indiscriminately. This secondary cutting activity is known as collateral cleavage.
To make this visible, scientists add “reporter” molecules to the mixture. These reporters consist of a light-emitting part and a quencher part that suppresses the light. When the Cas enzyme cuts these reporters, the light-emitting part separates from the quencher, releasing a detectable signal like a glow or color change.
Applications in Disease Detection
The adaptability of CRISPR diagnostics allows for its use across a wide spectrum of diseases. A primary application is identifying viral pathogens. The technology has been adapted to detect viruses such as SARS-CoV-2, Zika, and influenza by targeting their unique genetic signatures. This allows for swift diagnosis, which is useful during outbreaks.
The system is also effective for detecting bacterial infections. It can be programmed to identify the genetic material of bacteria like Mycobacterium tuberculosis, helping to diagnose infections quickly. It can also be used to detect genes that confer antibiotic resistance, providing information to guide treatment decisions.
In oncology, CRISPR diagnostics are used for “liquid biopsies.” These tests detect fragments of cancer-specific DNA (ctDNA) in a patient’s blood. This could lead to earlier cancer detection and a non-invasive way to monitor tumor response to therapy. The system’s high specificity allows it to distinguish between healthy and cancer-associated DNA.
Beyond infectious diseases and cancer, this technology is applied to identify genetic markers for inherited disorders. By designing guide RNAs that match specific gene variants or mutations, it is possible to screen for a range of genetic conditions. This demonstrates the platform’s potential to identify diverse biomarkers from various biological samples.
Advantages Over Traditional Diagnostic Methods
CRISPR-based diagnostics have several advantages over methods like Polymerase Chain Reaction (PCR). A primary benefit is speed and portability. While PCR requires hours and specialized lab equipment, many CRISPR assays deliver results in under an hour and can be used on simple formats like paper strips. This makes them well-suited for point-of-care use or in remote areas.
Another advantage is the reduced cost. The materials for CRISPR-based tests are inexpensive, making large-scale testing more financially accessible. Unlike PCR, many CRISPR methods work at a constant temperature (isothermal amplification), which eliminates the need for bulky and expensive equipment.
The accuracy of CRISPR systems is also a notable feature. The technology has high specificity, correctly identifying the intended genetic target without being confused by similar sequences. It also has high sensitivity, enabling it to detect very small amounts of a target sequence in a sample.
Notable CRISPR-Based Diagnostic Platforms
Two prominent platforms have demonstrated the power of CRISPR for diagnostics. The first is SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), which uses the Cas13 enzyme to recognize and detect specific RNA sequences, making it effective for identifying RNA viruses.
The second platform is DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter), which utilizes the Cas12a enzyme. Cas12a is programmed to find and detect DNA targets, making it suitable for diagnosing infections from DNA-based viruses like human papillomavirus (HPV) or identifying bacterial DNA.
The choice between platforms depends on the target molecule. SHERLOCK and its Cas13 enzyme are used for RNA targets, while DETECTR with its Cas12a enzyme is used for DNA targets.