Detection of CRISPR-dCas9 on DNA with Solid-State Nanopores
| Autor: | Jaco van der Torre, Cees Dekker, Anthony Birnie, Michel Bengtson, Laura Restrepo-Pérez, Stephanie J. Heerema, Wayne Yang |
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| Jazyk: | angličtina |
| Předmět: |
Letter
Bioengineering Sequence (biology) Biosensing Techniques 02 engineering and technology 010402 general chemistry 01 natural sciences DNA sequencing Nanopores chemistry.chemical_compound Genome editing diagnostics Humans CRISPR General Materials Science Binding site Binding Sites Chemistry Mechanical Engineering DNA General Chemistry 021001 nanoscience & nanotechnology Condensed Matter Physics 0104 chemical sciences Nanopore Biophysics biosensing CRISPR-Cas Systems CRISPR-Cas9 0210 nano-technology Biosensor RNA Guide Kinetoplastida |
| Zdroj: | Nano Letters Nano Letters: a journal dedicated to nanoscience and nanotechnology |
| ISSN: | 1530-6992 1530-6984 |
| DOI: | 10.1021/acs.nanolett.8b02968 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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