Amplification-Free, Single-Microbead-Based Cas12a Assay for One-Step DNA Detection at the Single-Molecule Level

Abstract

CRISPR/Cas-based systems are highly attractive for developing next-generation diagnostic technologies because of their intrinsic merits such as simplicity, sensitivity, and specificity. However, currently, nucleic acid amplification procedures are still needed to achieve attomolar sensitivity in most CRISPR/Cas-based assays, which causes high cost, operation difficulty, and low efficiency. Herein, we combine the CRISPR/Cas12a-based assay and a single-microbead detection platform for one-step and amplification-free detection of DNA at the single-molecule level. By modifying DNA reporters on a biomimetic membrane-coated microbead, the activated Cas12a by targets will cleave these reporters and lighten the bead within 10 min. The method allows the detection of the target down to three copies in a 5 μL sample. Furthermore, we successfully apply this method for the specific identification of viral infection, foodborne bacteria, and DNA mutation in real samples without extra nucleic acid amplification. We believe that this approach offers new insights for developing CRISPR/Cas-based DNA assays in biomedical applications.

CRISPR/Cas-Powered Amplification-Free Detection of Nucleic Acids: Current State of the Art, Challenges, and Futuristic Perspectives

“…(A) A single-microbead-based Cas12a assay for DNA detection. Reproduced with permission from ref . Copyright 2022 American Chemical Society.…”

“…Microbead-modified DNA reporters were used to establish an amplification-free detection method that could realize the one-step monitor of single-molecule limitation and remove amplification operations. 92 As shown in Figure 7A, the microbead was coated with lipid-membrane-modified DNA reporters of Cas12a, which were shredded because of the transcleavage to rapidly lighten the bead. The detection platform significantly reduced the complexity of operation and shortened the detection time from hours to minutes.…”

Sensitive Measurement of ALKBH3 Activity using a Dual DNA Amplifier by Coupling DNA Circuits with CRISPR/Cas12a

“…Because of the target DNA or RNA binding capacity and trans‐cleavage activity, CRISPR/Cas‐based diagnostic method, such as SHERLOCK [13,14] and DETERCTR [15] have been developed for nucleic acid detection with high selectivity and sensitivity. Among these, CRISPR/Cas12a have received much attention due to the fast kinetics, efficient signal amplification as well as the use of cost‐effective and stable DNA reporters [16–19] . CRISPR/Cas12a binds with a single‐strand guide RNA (crRNA) to recognize the target DNA and endows trans‐cleavage of non‐target single strand DNA, [20] providing a powerful platform for the detection and imaging of various biomolecules, including nucleic acid, [21] small molecules, [22] metal ions, [23] proteins, [24] and exosomes [25] .…”

“…Among these, CRISPR/Cas12a have received much attention due to the fast kinetics, efficient signal amplification as well as the use of cost-effective and stable DNA reporters. [16][17][18][19] CRISPR/Cas12a binds with a single-strand guide RNA (crRNA) to recognize the target DNA and endows transcleavage of non-target single strand DNA, [20] providing a powerful platform for the detection and imaging of various biomolecules, including nucleic acid, [21] small molecules, [22] metal ions, [23] proteins, [24] and exosomes. [25] Recently, CRISPR/ Cas12a system was also coupled with isothermal nucleic acid amplification technologies, such as enzyme-assisted amplification, [26][27] nonenzymatic DNA cascade circuits [28] and nanomaterials-based amplification [29] to improve the sensitivity.…”

Ultrasensitive miRNA Detection Based on Magnetic Upconversion Nanoparticle Enhancement and CRISPR/Cas13a-Driven Signal Amplification

“…In brief, the Cas13a protein assembles with crRNA and forms a Cas13a/crRNA complex to specifically recognize the target RNA, followed by activating its trans-cleavage activity to nonspecifically cleave the RNA molecules at a rate of ∼4854 turnovers per second. , Owing to its remarkable sensitivity and specificity, the CRISPR/Cas13a system shines in the field of molecular diagnostics. More and more researchers have developed various molecular detection methods combined with electrochemiluminescence, − fluorescence, − quantum dots, , and other nanomaterials , based on CRISPR/Cas13a. However, the applications of these techniques in the detection of complex biological samples are often limited on account of their characteristics such as photobleaching, instability, and high background signal of traditional fluorescent probes as well as high toxicity of quantum dots.…”