New CRISPR-based diagnostic test detects pathogens in blood without amplification

Bioengineering professor and The Grainger College of Engineering’s Dean, Rashid Bashir, led a team of researchers in a project that’s resulted in new technology that offers rapid, highly sensitive detection of multi-drug-resistant bacteria and other pathogens at low concentrations.

This research was featured in an article in the Proceedings of the National Academy of Sciences (PNAS).

Researchers designed a CRISPR-based test that rapidly detects low levels of pathogen genetic material in blood. This is done without the need for nucleic acid amplification.

In CRISPR/Cas-based diagnostic tests, guide RNAs bind pathogen DNA or RNA, triggering Cas enzymes to become active and cleave the reporter nucleic acids that fluoresce when cleaved. However, the single CRISPR-based technique does not detect pathogens at low levels without a preamplification step.

Bashir’s team created a CRISPR-based diagnostic test that bypasses that amplification step by combining two CRISPR/Cas units in a complex called CRISPR-Cascade. One unit contains a guide RNA specific for a pathogen nucleic acid of choice and a Cas protein. When the Cas cleaves specially engineered nucleic acids that are added to the system, parts of the nucleic acids are free to bind and activate a second CRISPR/Cas, triggering a positive feedback loop that results in a high signal-to-noise ratio.

The test demonstrated unprecedented sensitivity. It also detected multi-drug-resistant Staphylococcus aureus DNA without prior amplification at concentrations that were orders of magnitude lower than the limit of a test using a single Cas. The test provided a simple “yes/no” result for the presence of any one pathogen in samples spiked with four common bloodstream pathogens.

The researchers said these results could be used toward developing highly sensitive CRISPR-based diagnostic tests that can detect pathogens in minutes without nucleic acid amplification.