Journal
TALANTA
Volume 233, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.talanta.2021.122554
Keywords
CRISPR/Cas9; EXPAR; G-quadruplex; Naked-eye; ssRNA; ssDNA
Categories
Funding
- National Natural Science Foundation of China [81772290, 81271930]
- Graduate Scientific Research and Innovation Foundation of Chongqing, China [CYB20070]
- Fundamental Research Funds for the Central Universities [2020CDCGSW052, 2019CDYGZD007]
- Chongqing Municipal Training Program of Innovation and Entrepreneurship for Undergraduates [S201910611436]
- Strong-flavor Baijiu Solid-state Fermentation Key Laboratory of China light industry [2019JJ002]
- Chongqing Graduate Tutor Team Construction Project
- Chongqing University
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Cas-G4EX is a novel method for detecting single-stranded nucleic acids, combining the advantages of CRISPR/Cas9 and EXPAR, with outstanding site-specific recognition and high-performance amplification efficiency, applicable for the detection of any ssRNA or ssDNA. The method exhibits excellent discrimination for single-base mutation of single-stranded nucleic acids, providing a promising platform for applications in molecular diagnosis and pathological analysis.
Accurate and effective detection of single-stranded nucleic acids is vital in both disease diagnosis and patho-logical studies. Hence, we develop a PAMmer-assisted CRISPR/Cas9 system mediated G4-EXPAR (Cas-G4EX) strategy for site-specific detection of ssRNA and ssDNA. PAMmer-assisted CRISPR/Cas9 executes the site-specific cleavage of target ssRNA or ssDNA and released product fragment with the desired sequence at the 3'-terminal. This fragment serves as a primer to activate subsequent sequence-dependent exponential amplification reaction (EXPAR). The G-rich EXPAR products assembles with hemin to form a G-Quadruplex (G4/hemin). G4/hemin catalyzes ABTS-H2O2 system with the appearance of vivid green color, realizing naked-eye analysis. Cas-G4EX integrates the superiority of CRISPR/Cas9 and EXPAR, presenting outstanding site-specific recognition and high-performance amplification efficiency. Meanwhile, the programmability of CRISPR/Cas9 system makes the proposed method become a universal detection paradigm for any ssRNA or ssDNA. Cas-G4EX assay shows the linear relationship from 250 aM to 2.5 nM for ssRNA detection with the actual LOD of 250 aM, and that ranges from 100 aM to 1 nM for ssDNA detection with the actual LOD of 100 aM. Additionally, the acceptable recoveries of 101.48%-109.61% for ssRNA and 93.25%-111.98% for ssDNA in real detection of human serum are obtained for detection of single-strand nucleic acid in real samples. Cas-G4EX also exhibits the excellent discrimination for single-base mutation of single-stranded nucleic acids. Therefore, Cas-G4EX assay provides a promising platform in the applications of molecular diagnosis and pathological analysis.
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