CRISPR/Cas12a with Universal crRNA for Indiscriminate Virus Detection

Area of Science:

• Molecular Biology
• Biotechnology
• Infectious Disease Diagnostics

Background:

• Viruses pose significant public health challenges, necessitating accurate detection for diagnosis and infection control.
• CRISPR/Cas biosensors offer promising analytical performance, potentially augmenting traditional methods like RT-PCR for nucleic acid detection.

Purpose of the Study:

• To develop a versatile and sensitive CRISPR-Cas12a-based biosensor for indiscriminate virus detection.
• To overcome the target-dependence limitation of crRNA recognition in CRISPR-based diagnostics.

Main Methods:

• Utilized Cas12a with a universal crRNA and isothermal amplification for virus detection.
• Designed a system where amplified products trigger Cas12a/crRNA complex to cleave reporters, generating a signal.
• Demonstrated detection of multiple viral nucleic acids including SARS-CoV-2, HPV, HCOV-NL63, HCOV-HKU1, and miRNA biomarkers.

Main Results:

• The developed method achieved high sensitivity in detecting various viral nucleic acids.
• The system demonstrated versatility by detecting multiple viruses using the same crRNA.
• Successful detection of SARS-CoV-2 and HPV16 pseudoviruses validated the platform's diagnostic potential.

Conclusions:

• The proposed CRISPR-Cas12a biosensor is a versatile and sensitive platform for molecular diagnostics.
• This target-independent recognition strategy enhances the applicability of CRISPR-based virus detection.
• The method shows promise for broad-spectrum molecular diagnostic applications in public health.