期刊
BIOSENSORS & BIOELECTRONICS
卷 213, 期 -, 页码 -出版社
ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2022.114468
关键词
CRISPR-Cas; Biosensing; Histone deacetylase; Signal amplification; Deep learning
类别
资金
- National Key Research and Development Program of China [2020YFA0907500]
- National Natural Science Foundation of China [22034002, 21974038, 21725503]
The study demonstrates the use of a switchable Cas12a for direct analysis of histone deacetylase (HDAC) activity. The inactivation of Cas12a by acetylation can be reversed by HDAC-mediated deacetylation. By leveraging this switchable Cas12a, a sensitive one-pot assay for detecting HDAC activity was developed, with sub-nanomolar sensitivity. This assay can be applied in biochemical studies and clinical diagnostics.
The efficient and robust signal reporting ability of CRISPR-Cas system exhibits huge value in biosensing, but its applicability for non-nucleic acid analyte detection relies on the coupling of additional recognition modules. To address this limitation, we described a switchable Cas12a and exploited it for CRISPR-based direct analysis of histone deacetylase (HDAC) activity. Starting from the acetylation-mediated inactivation of Cas12a by antiCRISPR protein AcrVA5, we demonstrated that the acetyl-inactivated Cas12a could be reversibly activated by HDAC-mediated deacetylation based on computational simulations (e.g., deep learning and protein-protein docking analysis) and experimental verifications. By leveraging this switchable Cas12a for both target sensing and signal amplification, we established a sensitive one-pot assay capable of detecting deacetylase sirtuin-1 with sub-nanomolar sensitivity, which is 50 times lower than the standard two-step peptide-based assay. The versability of this assay was validated by the sensitive assessment of cellular HDAC activities in different cell lines with good accuracy, making it a valuable tool for biochemical studies and clinical diagnostics.
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