Nucleic Acid Detection through RNA-Guided Protease Activity in Type III-E CRISPR-Cas Systems

Area of Science:

• Molecular Biology
• Biotechnology
• CRISPR Systems

Background:

• Type III-E CRISPR-Cas systems (Craspase) exhibit RNA-guided protease activity.
• This offers a novel protein probe platform for nucleic acid detection, distinct from traditional nucleic acid probes.

Purpose of the Study:

• To develop and validate a Craspase-based assay for detecting specific single-stranded RNA (ssRNA) targets.
• To assess the assay's sensitivity, specificity, and multiplexing potential for viral pathogen detection.

Main Methods:

• Adapted a fluorescence readout assay using a dual-tagged protein substrate (Csx30) to monitor Craspase cleavage.
• Developed Craspase-based systems for detecting SARS-CoV-2, norovirus, and influenza virus (IFV) genes.
• Incorporated recombinase polymerase isothermal preamplification to enhance sensitivity.

Main Results:

• Demonstrated specific detection of target ssRNAs down to 25 pM.
• Achieved a detection limit of approximately 2 fM with preamplification, a ~15,000-fold improvement.
• Successfully performed multiplexed detection of multiple viral genes (SARS-CoV-2 spike and nsp12) and different viruses (SARS-CoV-2 and IFV).

Conclusions:

• The Craspase-based assay is a sensitive and specific platform for nucleic acid detection.
• Its ability to perform multiplexed diagnosis makes it adaptable for clinical applications.
• Further expansion with diverse fluorescent tags and Craspase systems can enhance its diagnostic utility.