Pathogens detected in blood at low concentrations by novel CRISPR test

Professor Rashid Bashir and a research team at the Grainger College of Engineering, Urbana, Illinois have developed a novel CRISPR-based method for the rapid detection of multi-drug-resistant bacteria with highly sensitive at low concentrations. The technology can identify low levels of pathogen genetic material in blood without the need for nucleic acid amplification.

In existing CRISPR/Cas-based diagnostic tests, guide RNAs bind to pathogen DNA or RNA, triggering Cas enzymes to become active and cleave the reporter nucleic acids so that they can fluoresce. However, the single CRISPR-based technique cannot detect pathogens at low levels without an amplification step.

Here Bashir’s team created a CRISPR-based diagnostic test that bypasses the need for amplification 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.

For further reading please visit: 10.1073/pnas.2420166122