The development of a sensitive, facile, and cost-effective colorimetric method is of great significance for the point-of-care testing of viral nucleic acid. Herein, we reported a strand displacement amplification assisted CRISPR-Cas12a (SDACC) method for the colorimetric analysis of viral nucleic acid. The hepatitis B virus (HBV) DNA was chosen as the target to trigger strand displacement amplification (SDA) and generate abundant single-strand DNA (ssDNA) products. The ssDNA amplicon hybridized with template DNA to activate the trans-cleavage activity of CRISPR-Cas12a, leading to the nonspecific cleavage of ssDNA on GOx-ssDNA-modified magnetic beads and the release of GOx. The released GOx was capable of catalyzing the substrate solution to generate a color change, which could be directly observed by naked eyes. The SDACC strategy could identify a single-base mismatch located in the DNA sequence and achieve a sensitive detection for HBV DNA with the limit of detection as low as 41.8 fM. Notably, the sophisticated primer design for target amplification and complicated detection process could be circumvented. The current approach realizes a simple, low-cost, and sensitive colorimetric detection for viral nucleic acid and holds great promise for the practical application of virus infection diagnosis.

Strand Displacement Amplification Assisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral Nucleic Acid.

Rapid and accurate detection of SARS-CoV-2 mutations using a Cas12a-based sensing platform

Besides gene-editing, the CRISPR/Cas12a system has also been widely used in in vitro biosensing, but its applications in live-cell biosensing are rare. One reason is lacking appropriate carriers to synchronously deliver all components of the CRISPR/Cas12a system into living cells. Herein, we demonstrate that MnO2 nanosheets are an excellent carrier of CRISPR/Cas12a due to the two important roles played by them. Through a simple mixing operation, all components of the CRISPR/Cas12a system can be loaded on MnO2 nanosheets and thus synchronously delivered into cells. Intracellular glutathione (GSH)-induced decomposition of MnO2 nanosheets not only results in the rapid release of the CRISPR/Cas12a system in cells but also provides Mn2+ as an accelerator to promote CRISPR/Cas12a-based biosensing of intracellular targets. Due to the merits of highly efficient delivery, rapid intracellular release, and the accelerated signal output reaction, MnO2 nanosheets work better than commercial liposome carriers in live-cell biosensing analysis of survivin messenger RNA (mRNA), producing much brighter fluorescence images in a shorter time. The use of MnO2 nanosheets might provide a good carrier for different CRISPR/Cas systems and achieve the rapid and sensitive live-cell biosensing analysis of different intracellular targets, thus paving a promising way to promote the applications of CRISPR/Cas systems in living cells.

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MnO2 nanosheets as a carrier and accelerator for improved live-cell biosensing application of CRISPR/Cas12a

In this work, boron and nitrogen-codoped carbon dots (BN-CDs) as highly efficient electrochemiluminescence (ECL) emitters with advantages of low excitation potential and high ECL efficiency were prepared to establish a novel ternary ECL system for ultrasensitive detection of HBV-DNA. Especially, both platinum nanoflowers (Pt NFs) and boron radicals (B•) from the BN-CDs could accelerate the reduction of coreactant S2O82- to abundant SO4•- simultaneously, making the BN-CDs have outstanding ECL performance. Impressively, the ECL efficiency of BN-CDs is much higher than that of nondoped CDs and single-doped CDs. In addition, by combining the novel ECL ternary system with the exonuclease III (Exo III)-induced target DNA amplification strategy, an ECL biosensor was constructed to realize the ultrasensitive detection of HBV-DNA from 100 aM to 1 nM, while the limit of detection was 18.08 aM. Therefore, a promising highly efficient ECL emitter was offered to develop a novel ECL detection method for clinical disease analysis. 

Boron and Nitrogen-Codoped Carbon Dots as Highly Efficient Electrochemiluminescence Emitters for Ultrasensitive Detection of Hepatitis B Virus DNA

Timely diagnosis is of great benefit to improve the survival rate of cancer patients. Body fluid cancer biomarker detection is a critical kind of noninvasive method for cancer diagnosis. Nevertheless, traditional methods for cancer biomarker detection always rely on a large-scale instrument and involve sophisticated operation. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-based in vitro diagnosis can simplify the detection procedures and improve sensitivity and specificity, holding great promise as the next-generation molecular diagnostic technology. In this Feature, we introduce the working mechanisms of different kinds of CRISPR/Cas systems for biosensing and CRISPR/Cas-mediated detection strategies for different kinds of cancer biomarkers including nucleic acids, proteins, and extracellular vesicles. In addition, the perspective and challenges of CRISPR/Cas-based strategies for cancer biomarkers are discussed.

Shiqi Zhang

Papers

CRISPR/Cas-Based In Vitro Diagnostic Platforms for Cancer Biomarker Detection.

Strand Displacement Amplification Assisted CRISPR-Cas12a Strategy for Colorimetric Analysis of Viral Nucleic Acid.