Nucleic Acid detection with Short Prokaryotic Argonautes (NASPA)

The SARS-CoV-2 pandemic highlighted the need for versatile diagnostics tools. Detecting presence of specific nucleic acid sequences is a powerful method for pathogen diagnostics, phenotyping, and determination of single nucleotide polymorphisms or other mutations underlying genetic diseases. PCR-based methods, the gold standard in nucleic acid detection, require specialized equipment and trained personnel, limiting its applicability at point-of-care or in-field locations. Alternative isothermal amplification/CRISPR-Cas-based detection methods suffer from technical limitations and a crowded patent landscape. Nucleic Acid detection with Short Prokaryotic Argonautes (NASPA) is a versatile, sensitive, and specific one-pot nucleic acid detection method that can be used for point-of-care or in-field diagnostics. NASPA builds on our research in which we characterized short prokaryotic Argonaute (pAgo) systems. In NASPA, short pAgos are programmed with synthetic 15-21 nt guide RNAs to detect complementary DNA sequences. Upon detection of the DNA, breakdown of reporter ε-NAD+ is triggered, resulting in a traceable fluorescent signal. As there are no sequence requirements for the guide RNA, and guide RNAs can readily be synthesized, NASPA facilitates detection of virtually any sequence of choice. We have proof of concept for NASPA-based diagnostics in a laboratory setting (technology readiness level (TRL) 3), and intellectual property has been protected through patent applications. The proposed ERC PoC project has two main objectives 1) enhance NASPA and enable detection of RNA and DNA sequences of economic and/or medical relevance in point-of-care and in-field settings, reaching TLR 5, and 2) build a network of stakeholders including academic and commercial partners to expedite the development of NASPA-based applications and establish a valorisation strategy. Achieving these objectives will pave the way for further development and valorisation of NASPA-based diagnostics.