CAS10-BASED ASSAY FOR NUCLEIC ACID DETECTION

Smart overview of the Invention

A novel detection system utilizes a CRISPR-associated effector protein, specifically Cas10, which is programmed to bind target nucleic acids through complementary base pairing. Upon binding to RNA, Cas10 generates cyclic oligoadenylate (cOA), activating a nuclease such as NucC. This activation leads to a reporter system producing a detectable fluorescent or colored signal, facilitating the identification of target nucleic acids in samples.

Background on Viral Detection

Infectious agents like SARS-COV-2 pose significant public health challenges. Effective detection technologies are essential for combating these threats, with ideal tests being specific, sensitive, rapid, scalable, and cost-effective. Traditional RT-PCR methods convert RNA to DNA for amplification, requiring complex equipment and taking 1-2 hours. Alternative isothermal amplification techniques have emerged but still rely on RNA conversion to DNA.

CRISPR-Based Detection Advantages

This detection system leverages the Cas10 protein from Type III CRISPR systems to detect specific RNA sequences. The binding of a target nucleic acid activates Cas10, which generates cOA in large quantities for each detected RNA molecule. This approach allows customization for various target nucleic acids and enhances sensitivity, making it suitable for diagnosing diseases caused by viral pathogens like SARS-COV-2.

Methodology and Applications

• The system includes Cas10, a binding molecule for the target nucleic acid, a nuclease, and a reporter system.
• Upon interaction with a sample, if the target nucleic acid is present, the binding event activates the system, leading to signal generation.
• In contrast, if the target is absent, no signal is produced.

Limit of Detection and Target Specificity

The disclosed systems enable detection of very low amounts of target nucleic acid, with limits of detection potentially below 1 fM. The method can be applied to various RNA types, including those from pathogens like SARS-COV-2 or other viruses. Target sequences can be derived from specific genes within viral genomes, enhancing the system's utility in diagnosing related diseases effectively.