CRISPR/Cas12a-Mediated Interfacial Cleaving of Hairpin DNA Reporter for Electrochemical Nucleic Acid Sensing

Abstract

A rapid and sensitive isothermal method is crucial for point-of-care (POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins were discovered to exhibit target-triggered nonspecific single-stranded deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA) and linear DNA was investigated in detailed. A novel electrochemical DNA biosensor was then developed via target-induced Cas12a cleaving interfacial hpDNA. In this strategy, the RNA-guided target DNA binding activates the robust Cas12a ssDNase activity. The immobilized hpDNA electrochemical reporters with a low surface coverage and incompact morphological structure present accessible substrates for highly efficient Cas12a cleavage, leading to a highly sensitive electrochemical DNA biosensor. Under the optimal conditions, as low as 30 pM target DNA was detected in about 60 min with 3.5 orders of magnitude dynamic range from 50 pM to 100 nM. Furthermore, the practical application ability of the established sensing method for detecting the target in complex matrices was also demonstrated. The proposed strategy enables rapid and sensitive DNA determination, providing a potential tool for POC molecular diagnostics.

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

“…Several sensor types have been created by researchers so far, in which no target-amplification is needed to measure at a very low LOD. These sensors are a graphene-based field effect transistor (gFET) ( Hajian et al, 2019 ), nanopore sensors ( Weckman et al, 2019 ; Yang et al, 2018 ), electrochemical sensors ( Bruch et al, 2019 ; Dai et al, 2019 ; Xu et al, 2020 ; D. Zhang et al, 2020 ) and a conductivity sensor combined with a DNA gel ( English et al, 2019 ).…”

“…7 An overview of the mechanisms in the different electronic sensors described in the literature. A) gFET ( Hajian et al, 2019 ), B) nanopore sensor, adjusted from ( Yang et al, 2018 ), C) different types of electrochemical sensors with MB as an electrochemical tag, based on targeted (II) and collateral cleavage (I & III) ( Dai et al, 2019 ; Xu et al, 2020 ; D. Zhang et al, 2020 ) D) collateral cleavage based sensor with a glucose oxidase electrochemical tag. Adapted from ( Bruch et al, 2019 ), according to Creative Commons Attribution License (CC BY 4.0) E) collateral cleavage of DNA-gel based sensor with combined colorimetric and amperometric readout ( English et al, 2019 ).…”

“…Zhang et al used the same approach as Xu et al to increase the sensitivity of the system by Dai et al, with MB molecules brought close to a gold surface by hairpin DNA ( Zhang et al, 2020 ) ( Fig. 7 C–II).…”

Recent Improvements in CRISPR-Based Amplification-Free Pathogen Detection

“…E-CRISPR successfully detects target nucleic acids such as human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19) at the picomolar level without preamplification of target DNA. The ssDNA reporter can also be optimized to the interfacial hairpin DNA that has an incompact morphological structure to facilitate highly efficient Cas12a cleavage, leading to higher detection sensitivity ( Zhang et al, 2020 ). Using the same electrochemical strategy, Cas12a has been used in direct detection of many other pathogens such as the dengue virus with the LOD of 100 fM ( Lee et al, 2021 ).…”

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas Advancement in Molecular Diagnostics and Signal Readout Approaches

“…This system was used in the detection of human papillomavirus 16 and parvovirus B19 with the limit of detection (LOD) of 50 pmol/L. 20 The method sensitivity was further improved by Zhang et al 21 by replacing ssDNA reporter with a hairpin DNA reporter. Because of the shortening of electron tunneling distance between the electrode surface and MB dye, enhanced signal change was achieved.…”