TY - JOUR
T1 - Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores
AU - Yang, Wayne
AU - Restrepo-Pérez, Laura
AU - Bengtson, Michel
AU - Heerema, Stephanie J.
AU - Birnie, Anthony
AU - Van Der Torre, Jaco
AU - Dekker, Cees
PY - 2018
Y1 - 2018
N2 - 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.
AB - 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.
KW - biosensing
KW - CRISPR-Cas9
KW - diagnostics
KW - Nanopores
UR - http://resolver.tudelft.nl/uuid:d0d99406-5b8f-4292-89c0-cf02de707903
UR - http://www.scopus.com/inward/record.url?scp=85053617399&partnerID=8YFLogxK
U2 - 10.1021/acs.nanolett.8b02968
DO - 10.1021/acs.nanolett.8b02968
M3 - Article
AN - SCOPUS:85053617399
JO - Nano Letters: a journal dedicated to nanoscience and nanotechnology
JF - Nano Letters: a journal dedicated to nanoscience and nanotechnology
SN - 1530-6984
ER -