Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores

Abstract

Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.

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

“…Using nanopores as a sensing strategy enables detection of dsDNA without unzipping or temperature changes and also opens up opportunities for multiplexing. Yang et al showed that the binding position of the dCas9 effector protein could be deduced from the ionic current spike, which was later also confirmed by Weckman et al ( Weckman et al, 2019 ; Yang et al, 2018 ). The latter authors designed multiple crRNA sequences for dCas9 that could all bind to the same DNA sequence, creating ‘barcodes’ in the ionic current traces specific for the different DNA sequences.…”

“…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 ).…”

Comparing current noise in biological and solid-state nanopores

“…Nanopores are promising tools for biosensing applications and sequencing of DNA and proteins, as they can resolve single analyte molecules, resolve structural modifications of molecules, and even discriminate between nucleotide sequences [1][2][3][4][5][6][7][8][9][10] . The detection mechanism is simple: while passing through the pore, a (part of a) molecule transiently blocks the ionic current, thereby inducing a small dip in the current signal, which is detectable by the electronics (Fig.…”

Engineering and Modeling the Electrophoretic Trapping of a Single Protein Inside a Nanopore

“…38 Electrophoretic translocation of protein–DNA complexes through small nanopores (<3 nm diameter) typically results in the temporary trapping of the entire complex, which has allowed for the study of polymerase enzymes 39,40 and DNA-binding proteins. 41,42 Although promising, none of these approaches could efficiently control the trapping of the protein inside the nanopore or allow observation of enzyme kinetics or ligand-induced conformational changes.…”