tetano
Editor, Senior Moderator
Anal Biochem
. 2023 Jan 11;115046.
doi: 10.1016/j.ab.2023.115046. Online ahead of print.
PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA
Kai Shi[SUP] 1 [/SUP], Zhigang Yi[SUP] 2 [/SUP], Yaoxia Han[SUP] 2 [/SUP], Jiaxuan Chen[SUP] 2 [/SUP], Yu Hu[SUP] 2 [/SUP], Ying Cheng[SUP] 2 [/SUP], Sujun Liu[SUP] 2 [/SUP], Wei Wang[SUP] 2 [/SUP], Jiuhua Song[SUP] 3 [/SUP]
Affiliations
Abstract
The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reactions (TSDRs) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDRs, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDRs, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.
Keywords: CRISPR-Cas12a; Electrochemical biosensor; PAM-Free TSDR; SARS-CoV-2.
. 2023 Jan 11;115046.
doi: 10.1016/j.ab.2023.115046. Online ahead of print.
PAM-free cascaded strand displacement coupled with CRISPR-Cas12a for amplified electrochemical detection of SARS-CoV-2 RNA
Kai Shi[SUP] 1 [/SUP], Zhigang Yi[SUP] 2 [/SUP], Yaoxia Han[SUP] 2 [/SUP], Jiaxuan Chen[SUP] 2 [/SUP], Yu Hu[SUP] 2 [/SUP], Ying Cheng[SUP] 2 [/SUP], Sujun Liu[SUP] 2 [/SUP], Wei Wang[SUP] 2 [/SUP], Jiuhua Song[SUP] 3 [/SUP]
Affiliations
- PMID: 36641031
- DOI: 10.1016/j.ab.2023.115046
Abstract
The early diagnosis of coronavirus disease 2019 (COVID-19) is dependent on the specific and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Herein, we develop a highly sensitive and specific electrochemical biosensor for SARS-CoV-2 target RNA detection based on the integration of protospacer adjacent motif (PAM)-free cascaded toehold-mediated strand displacement reactions (TSDRs) and CRISPR-Cas12a (PfTSDR-CRISPR). In this study, each target is transformed into multiple DNA substrates with bubble structure in the seed region by the cascaded TSDRs, which can directly hybridize with guide RNA (gRNA) without PAM requirement and then activate CRISPR-Cas12a's trans-cleavage activity. Subsequently, the hairpin DNA modified with methylene blue (MB-HP) is cleaved by activated CRISPR-Cas12a. Therefore, as MB leaves the electrode surface, a decreased current signal is obtained. With the involvement of PAM-free cascaded TSDRs and CRISPR-Cas12a amplification strategy, the PfTSDR-CRISPR-based electrochemical biosensor achieves the detection of target RNA as low as 40 aM. The biosensor has high sequence specificity, reliability and robustness. Thanks to the PAM-free cascaded TSDRs, the biosensor can achieve universal detection of different target RNA without redesigning gRNA sequence of CRISPR-Cas12a. In addition, this biosensor successfully detects SARS-CoV-2 target RNA in complex samples, which highlights its potential for diagnosing COVID-19.
Keywords: CRISPR-Cas12a; Electrochemical biosensor; PAM-Free TSDR; SARS-CoV-2.