Photocontrolled crRNA activation enables robust CRISPR-Cas12a diagnostics.
Menglu HuZhiqiang QiuZirong BiTian TianYongzhong JiangXiaoming ZhouPublished in: Proceedings of the National Academy of Sciences of the United States of America (2022)
CRISPR diagnostics based on nucleic acid amplification faces barriers to its commercial use, such as contamination risks and insufficient sensitivity. Here, we propose a robust solution involving optochemical control of CRISPR RNA (crRNA) activation in CRISPR detection. Based on this strategy, recombinase polymerase amplification (RPA) and CRISPR-Cas12a detection systems can be integrated into a completely closed test tube. crRNA can be designed to be temporarily inactivated so that RPA is not affected by Cas12a cleavage. After the RPA reaction is completed, the CRISPR-Cas12a detection system is activated under rapid light irradiation. This photocontrolled, fully closed CRISPR diagnostic system avoids contamination risks and exhibits a more than two orders of magnitude improvement in sensitivity compared with the conventional one-pot assay. This photocontrolled CRISPR method was applied to the clinical detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, achieving detection sensitivity and specificity comparable to those of PCR. Furthermore, a compact and automatic photocontrolled CRISPR detection device was constructed.
Keyphrases
- crispr cas
- genome editing
- loop mediated isothermal amplification
- nucleic acid
- sars cov
- label free
- real time pcr
- genome wide
- respiratory syndrome coronavirus
- risk assessment
- gene expression
- drinking water
- coronavirus disease
- climate change
- radiation therapy
- deep learning
- sensitive detection
- wastewater treatment
- high throughput
- health risk
- transcription factor
- dna binding