A Simple and Highly Sensitive Naked-Eye Analysis of EGFR 19del via CRISPR/Cas12a Triggered No-Nonspecific Nucleic Acid Amplification

Abstract

The mutation status of epidermal growth factor receptor (EGFR) exon 19 is of great importance for predicting sensitivity to tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). However, the development of simple, sensitive, and no-nonspecific amplification platforms for EGFR 19del detection in NSCLC remains a challenge. Herein, we developed a novel, simple, and highly sensitive naked-eye assay utilizing CRISPR/Cas12a-triggered no-nonspecific nucleic acid amplification (NAA) with rolling circle amplification (RCA) as a model for EGFR 19del detection. Typically, circular padlocks are designed to be the trans-cleavage substrate of Cas12a/crRNA and serve as templates for RCA. Since the target EGFR 19del induces robust trans-cleavage activity of the Cas12a/crRNA duplex, the surrounding circular padlocks are cleaved into random short linear fragments that are unable to initiate RCA, resulting in a colorless solution. However, in the absence of EGFR 19del, the inactivated Cas12a enzymes cannot cleave the circular padlocks, and they remain able to serve as templates to initiate RCA to generate long single-stranded DNA to further fold into G-quadruplex/hemin DNAzymes to catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2− ), generating a color response that is obvious to the naked eye. As expected, this strategy with a detection limit as low as 20 fM exhibited robust selectivity and anti-interference ability. Moreover, this method was applicable for detecting EGFR 19del in real serum samples and showed high consistency with real-time quantitative polymerase chain reaction (qPCR) and sequencing results, providing a promising strategy for the early noninvasive diagnosis and guidance of clinical treatment for cancer.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection

“…In comparison with other nucleic acid detection assays, as revealed in Table S4, the LCR-CPDzyme system exhibits the advantages of being cost-effective and having high sensitivity. In particular, the sensitivity of this study is much higher than that of other biosensing platforms by using G4/hemin. − …”

Endonuclease-Assisted PAM-free Recombinase Polymerase Amplification Coupling with CRISPR/Cas12a (E-PfRPA/Cas) for Sensitive Detection of DNA Methylation

“…By combining nucleic acid amplification with the CRISPR/Cas system, the detection sensitivity and specificity are significantly improved. In addition, a variety of techniques are developed based on different CRISPR/Cas systems, such as SHERLOCK (Cas13), , DETECTR (Cas12 or Cas14), , and HOLMES (Cas12). , In the V-A type CRISPR system, CRISPR/Cas12a (Cpf1) exhibits specific recognition ability for double-stranded DNA (dsDNA) and can stimulate its trans-cleavage activity for target detection . However, these usually require the protospacer adjacent motif (PAM) sites to locate the target region.…”

“…29,30 In the V-A type CRISPR system, CRISPR/Cas12a (Cpf1) exhibits specific recognition ability for double-stranded DNA (dsDNA) and can stimulate its trans-cleavage activity for target detection. 31 However, these usually require the protospacer adjacent motif (PAM) sites to locate the target region. Actually, many disease-related targets have no PAM sites, which greatly limits the application of the CRISPR/Cas system.…”

Sensitive and Amplification-Free Electrochemiluminescence Biosensor for HPV-16 Detection Based on CRISPR/Cas12a and DNA Tetrahedron Nanostructures

“…One is to use the trans-cleavage property of CRISPR/Cas12 system to cleave the initiation chain of the amplification reaction, such as the hybridization chain reaction (HCR) or rolling circle amplification (RCA), and inhibit the reaction process. 34,35 Another commonly used strategy is to transform the tested substance to produce more activation chains (crRNA or target of CRISPR/Cas12 system) to improve the sensitivity of the CRISPR/Cas12 system through a nucleic acid amplification strategy. 36−38 However, most of these strategies are based on enzyme-assisted amplification, which takes a segment of DNA as a template to form the new activation chain by deoxyribonucleoside triphosphate (dNTPs) with the help of polymerase.…”

“…In recent years, the nucleic acid amplification techniques have been integrated with CRISPR/Cas12 to construct ECL sensors. − Currently, the reported amplification strategies based on the CRISPR/Cas12 system are mainly divided into two types. One is to use the trans-cleavage property of CRISPR/Cas12 system to cleave the initiation chain of the amplification reaction, such as the hybridization chain reaction (HCR) or rolling circle amplification (RCA), and inhibit the reaction process. , Another commonly used strategy is to transform the tested substance to produce more activation chains (crRNA or target of CRISPR/Cas12 system) to improve the sensitivity of the CRISPR/Cas12 system through a nucleic acid amplification strategy. − However, most of these strategies are based on enzyme-assisted amplification, which takes a segment of DNA as a template to form the new activation chain by deoxyribonucleoside triphosphate (dNTPs) with the help of polymerase. , This is because if the activation strand exists in the original nucleic acid, the original nucleic acid may activate Cas12a to produce leakage. In addition, the non-specific trans-cleavage property of Cas12a also splices original single-stranded nucleic acid.…”