1. Efficient, specific and direct detection of double-stranded DNA targets using Cas12f1 nucleases and engineered guide RNAs.
- Author
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He J, Hu X, Weng X, Wang H, Yu J, Jiang T, Zou L, Zhou X, Lyu Z, Liu J, Zhou P, Xiao X, Zhen D, and Deng Z
- Subjects
- Humans, Hepatitis B virus genetics, Hepatitis B virus isolation & purification, DNA genetics, DNA chemistry, Mycoplasma pneumoniae genetics, Mycoplasma pneumoniae isolation & purification, CRISPR-Associated Proteins genetics, Carcinoma, Non-Small-Cell Lung genetics, Carcinoma, Non-Small-Cell Lung diagnosis, Bacterial Proteins genetics, Bacterial Proteins chemistry, Lung Neoplasms genetics, Lung Neoplasms diagnosis, Endodeoxyribonucleases genetics, Endodeoxyribonucleases chemistry, Pneumonia, Mycoplasma diagnosis, Biosensing Techniques methods, CRISPR-Cas Systems, RNA, Guide, CRISPR-Cas Systems genetics
- Abstract
To address the limitations of the CRISPR/Cas12f1 system in clinical diagnostics, which require the complex preparation of single-stranded DNA (ssDNA) or in vitro transcripts (RNA), we developed a fluorescent biosensor named PDTCTR (PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter). This innovative biosensor integrates Recombinase Polymerase Amplification (RPA) with the Cas12f_ge4.1 system, facilitating the direct detection of double-stranded DNA (dsDNA). PDTCTR represents a significant leap forward, exhibiting a detection sensitivity that is a hundredfold greater than the original Cas12f1 system. It demonstrates the capability to detect Mycoplasma pneumoniae (M. pneumoniae) and Hepatitis B virus (HBV) with excellent sensitivity of 10 copies per microliter (16.8 aM) and distinguishes single nucleotide variations (SNVs) with high precision, including the EGFR (L858R) mutations prevalent in non-small cell lung cancer (NSCLC). Clinical evaluations of PDTCTR have demonstrated its high sensitivity and specificity, with rates ranging from 93%-100% and 100%, respectively, highlighting its potential to revolutionize diagnostic approaches for infectious diseases and cancer-related SNVs.This research underscores the substantial advancements in CRISPR technology for clinical diagnostics and its promising future in early disease detection and personalized medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)
- Published
- 2024
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