Key result
A CRISPR/Cas12a biosensor utilizing a universal crRNA and isothermal amplification successfully detected multiple viral nucleic acids, including SARS-CoV-2 and HPV, with a sensitivity as low as 5 pM.
A novel CRISPR/Cas12a biosensor with universal crRNA enables highly sensitive and versatile detection of multiple viral nucleic acids, potentially augmenting RT-PCR.
CRISPR/Cas biosensors may augment RT-PCR for virus detection; leaves open clinical adoption pending rigorous validation.
Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to SARS-CoV-2, human papillomaviruses (HPV), HCOV-NL63, HCOV-HKU1, and miRNA biomarkers. Taking SARS-CoV-2 and HPV16 pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications.
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Shang et al. (2024) studied Viral infection (SARS-CoV-2, HPV, etc.). CRISPR/Cas12a with universal crRNA and isothermal amplification vs. No Template Control (NTC) was evaluated on Limit of detection (sensitivity) for viral nucleic acids. A CRISPR/Cas12a biosensor utilizing a universal crRNA and isothermal amplification successfully detected multiple viral nucleic acids, including SARS-CoV-2 and HPV, with a sensitivity as low as 5 pM.
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