Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection

Fengqin Li; Junru Li; Weiqiang Yang; Shuai Yang; Congshuo Chen; Ling Du; Junyang Mei; Qianyun Tang; Xiaojing Chen; Chi Yao; Dayong Yang; Xiaolei Zuo; Peifeng Liu

doi:10.1002/anie.202305536

Framework-Hotspot Enhanced Trans Cleavage of CRISPR-Cas12a for Clinical Samples Detection

Angew Chem Int Ed Engl. 2023 Aug 7;62(32):e202305536. doi: 10.1002/anie.202305536. Epub 2023 Jun 27.

Authors

Fengqin Li^{1

2}, Junru Li^{1

2}, Weiqiang Yang³, Shuai Yang^{1

2}, Congshuo Chen^{1

2}, Ling Du^{1

2}, Junyang Mei^{1

2}, Qianyun Tang^{1

2}, Xiaojing Chen^{1

2}, Chi Yao⁴, Dayong Yang⁴, Xiaolei Zuo⁵, Peifeng Liu^{1

2}

Affiliations

¹ State Key Laboratory of Systems Medicine for Cancer, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200032, P. R. China.
² Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, P. R. China.
³ Emergency Department, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, P. R. China.
⁴ Frontiers Science Center for Synthetic Biology, Key Laboratory of Systems Bioengineering (MOE), Institute of Biomolecular and Biomedical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300350, P. R. China.
⁵ Institute of Molecular Medicine, Shanghai Key Laboratory for Nucleic, Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, P. R. China.

PMID: 37278518
DOI: 10.1002/anie.202305536

Abstract

The trans-cleavage property of CRISPR-Cas12a system makes it an excellent tool for disease diagnosis. Nevertheless, most methods based on CRISPR-Cas system still require pre-amplification of the target to achieve the desired detection sensitivity. Here we generate Framework-Hotspot reporters (FHRs) with different local densities to investigate their effect on trans-cleavage activity of Cas12a. We find that the cleavage efficiency increases and the cleavage rate accelerates with increasing reporter density. We further construct a modular sensing platform with CRISPR-Cas12a-based target recognition and FHR-based signal transduction. Encouragingly, this modular platform enables sensitive (100 fM) and rapid (<15 min) detection of pathogen nucleic acids without pre-amplification, as well as detection of tumor protein markers in clinical samples. The design provides a facile strategy for enhanced trans cleavage of Cas12a, which accelerates and broadens its applications in biosensing.

Keywords: Biomarkers; CRISPR-Cas12a; SARS-CoV-2; Tetrahedral DNA Framework; Trans-Cleavage.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biomarkers, Tumor
Biosensing Techniques*
CRISPR-Cas Systems / genetics
Nucleic Acids*
Signal Transduction

Substances

Biomarkers, Tumor
Nucleic Acids