Construction of a CRISPR‐Biolayer Interferometry Platform for Real‐Time, Sensitive, and Specific DNA Detection

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has been widely applied for nucleic acid detection because of its high specificity. By using the highly specific and irreversible bond between HaloTag and its alkane chlorine ligand, we modified dCas9 (deactivated CRISPR/Cas9) with biotin as a biosensor to detect nucleic acids. The CRISPR biosensor was facilely prepared to adequately maintain its DNA‐recognition capability. Furthermore, by coupling biolayer interferometry (BLI) with the CRISPR biosensor, a real‐time, sensitive, and rapid digital system called CRISPR‐BLI was established for the detection of double‐stranded DNA. The CRISPR biosensor immobilised on the biolayer could recruit the target DNA onto the biosensor surface and change its optical thickness, resulting in a shift in the interference pattern and responding signal of the BLI. The CRISPR‐BLI system was further applied to detect the ALP gene of Escherichia coli DH5α combined with a polymerase chain reaction, which demonstrated a linear range from 20 to 20 000 pg and a low detection limit (1.34 pg). The CRISPR‐BLI system is a promising approach for rapid and sensitive detection of target DNA analytes.

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…Additionally, a range of platforms have been developed by leveraging the advantages of CRISPR‐Cas with nucleic acid amplification technologies such as PCR (Ma, Peng, et al., 2021), RAA (Li, Ye, Chen, Xiang, et al., 2021), RPA (Chen et al., 2020; Zhuang et al., 2022), LAMP (Li, Chen, et al., 2022), multiple cross‐displacement amplification (Zhu et al., 2021), strand displacement amplification (Wang, Liu, et al., 2019; Zhou et al., 2018), rolling circle amplification (Qing et al., 2021), hybridization chain reaction (Liu et al., 2022), exponential amplification method (Huang et al., 2018; Tian et al., 2020), nicking enzyme‐assisted amplification (Bai et al., 2022), and so on. The CRISPR‐Cas biosensors are usually based on fluorescence signal readouts; other signal readouts such as colorimetry (Li, Zheng, et al., 2021), electrochemistry (Qing et al., 2021), lateral flow assay (Marsic et al., 2021), photothermal effect (Ma, Peng, et al., 2021), portable personal glucose meter (Liu, Hu, et al., 2021), surface‐enhanced Raman scattering (SERS) assay (Kim, Lee, Seo, et al., 2020; Pan et al., 2022), gas bubble signal (Silva et al., 2021), microfluidic paper‐based analytical device (μPAD) (Zhuang et al., 2022), hydrogel‐integrated paper‐based analytical device (μReaCH‐PAD) (Huang, Ni, et al., 2021), coupling biolayer interferometry (Qiao, Liu, et al., 2021), luminescence resonance energy transfer (Lin et al., 2022), and so on are established for visual and rapid biosensing applications.…”

Solid-Phase Collateral Cleavage System Based on CRISPR/Cas12 and Its Application toward Facile One-Pot Multiplex Double-Stranded DNA Detection

“…In recent years, the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system has drawn attention as an alternative nucleic acid sequence recognition tool for ssDNA–ssDNA hybridization. Considering that the Cas9 and CRISPR RNA (crRNA) complex can directly hybridize with target dsDNA via the complementary sequence to the crRNA, , the application of CRISPR/Cas9 has been extended to not only gene editing but also nucleic acid detection. − In previous reports, target dsDNA was captured by Cas9-crRNA immobilized on a sensor chip, and negative charge, change in electrical resistance, and the thickness of the captured dsDNA were detected by a graphene-based field effect transistor, , a screen-printed electrode, and a biolayer interferometry detector, respectively. In addition, the colorimetric detection using Cas9-crRNA as an alternative to primary antibodies in enzyme-linked immunosorbent assays (ELISAs) has also been reported …”