Sequence-Specific Recognition of HIV-1 DNA with Solid-State CRISPR-Cas12a-Assisted Nanopores (SCAN)

Abstract

Nucleic acid detection methods are crucial for many fields such as pathogen detection and genotyping. Solid-state nanopore sensors represent a promising platform for nucleic acid detection due to its unique single molecule sensitivity and label-free electronic sensing. Here, we demonstrated the use of the glass nanopore for highly sensitive quantification of single-stranded circular DNAs (reporters), which could be degraded under the trans-cleavage activity of the target-specific CRISPR-Cas12a. We developed and optimized the Cas12a assay for HIV-1 analysis. We validated the concept of the solid-state CRISPR-Cas12a-assisted nanopores (SCAN) to specifically detect the HIV-1 DNAs. We showed that the glass nanopore sensor is effective in monitoring the cleavage activity of the target DNA-activated Cas12a. We developed a model to predict the total experimental time needed for making a statistically confident positive/negative call in a qualitative test. The SCAN concept combines the much-needed specificity and sensitivity into a single platform, and we anticipate that the SCAN would provide a compact, rapid, and low-cost method for nucleic acid detection at the point of care.

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

“…While nanopore sensing could be useful for identifying DNA, it is low throughput, and therefore a very slow method for low LOD measurements. A more recent publication by Nouri et al potentially solved this measurement time issue of low concentrations of target DNA exploiteding the trans -cleavage of Cas12a and measuring the occurrence of reporter DNA fragments ( Nouri et al, 2020 ).…”

Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

“…Besides, the solid-state nanopore sensor has shown great potential in detecting single molecules and is an alternative approach to magnify the signal output. With the combination of Cas12a-mediated trans -cleavage and the glass nanopore sensor toward an electronic sensing platform, target nucleic acids such as the HIV-1 DNA can be specifically and sensitively detected without the requirement of preamplification ( Nouri et al, 2020 ).…”

CRISPR detectives against SARS-CoV-2: a major setback against COVID-19 blowout

“…CRISPR-based sensing has exhibited its great potential in specific, sensitive, and affordable detection of nucleic acid in the last couple of years, and presently SARS-CoV-2, but still this arena needs much more attention for overcoming the challenges associated with this technology for the development of “sample-in-answer-out” CRISPR-based point of care devices. According to the WHO guidelines, the key features of a point of care diagnostic device are affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable (ASSURED) (Nouri et al 2020 ). Abiding by these guidelines, the assay runtime of the CRISPR-based methods is one of the most crucial drawback, since sample preparation involves the most part of the CRISPR-based diagnostic methods, as well as separate sample preparations also increases the complexity of the process and in turn reduces the acceptability and applicability (Lino et al 2018 ; Abbott et al 2020 ; Nouri et al 2020 ).…”

“…According to the WHO guidelines, the key features of a point of care diagnostic device are affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable (ASSURED) (Nouri et al 2020 ). Abiding by these guidelines, the assay runtime of the CRISPR-based methods is one of the most crucial drawback, since sample preparation involves the most part of the CRISPR-based diagnostic methods, as well as separate sample preparations also increases the complexity of the process and in turn reduces the acceptability and applicability (Lino et al 2018 ; Abbott et al 2020 ; Nouri et al 2020 ). This necessitates the development of a single-step diagnostic test for efficient and rapid point of care application.…”