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CRISPR-based DNA and RNA detection with liquid-liquid phase separation

View ORCID ProfileWillem Kasper Spoelstra, Jeroen M. Jacques, View ORCID ProfileFranklin L. Nobrega, Anna C. Haagsma, Marileen Dogterom, View ORCID ProfileTimon Idema, View ORCID ProfileStan J. J. Brouns, View ORCID ProfileLouis Reese
doi: https://doi.org/10.1101/471482
Willem Kasper Spoelstra
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Jeroen M. Jacques
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Franklin L. Nobrega
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Anna C. Haagsma
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Marileen Dogterom
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Timon Idema
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Stan J. J. Brouns
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
2Laboratory of Microbiology, Wageningen University, Stippeneng 4, 6708 WE Wageningen, The Netherlands
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  • For correspondence: stanbrouns@gmail.com
Louis Reese
1Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands
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Abstract

The ability to detect specific nucleic acid sequences allows for a wide range of applications including identification of pathogens, clinical diagnostics, and genotyping. CRISPR-Cas proteins Cas12a and Cas13a are RNA-guided endonucleases that bind and cleave specific DNA and RNA sequences, respectively. After recognition of a target sequence both enzymes activate a unique, indiscriminate nucleic acid cleavage activity, which has been exploited for detection of sequence specific nucleotides using labelled reporter molecules. We here present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Turbidity arises from coacervates of positively charged polyelectrolytes with long poly(dT) or poly(U) oligonucleotides. In the presence of a target sequence, long oligonucleotides are progressively shortened, changing the solution from turbid to transparent. We explain how oligonucleotide cleavage resolves LLPS by using a mathematical model which we validate with poly(dT) phase separation experiments. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

Footnotes

  • Results and discussion sections updated and extended; Figures rearranged and revised; additional references added

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 15, 2019.
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CRISPR-based DNA and RNA detection with liquid-liquid phase separation
Willem Kasper Spoelstra, Jeroen M. Jacques, Franklin L. Nobrega, Anna C. Haagsma, Marileen Dogterom, Timon Idema, Stan J. J. Brouns, Louis Reese
bioRxiv 471482; doi: https://doi.org/10.1101/471482
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CRISPR-based DNA and RNA detection with liquid-liquid phase separation
Willem Kasper Spoelstra, Jeroen M. Jacques, Franklin L. Nobrega, Anna C. Haagsma, Marileen Dogterom, Timon Idema, Stan J. J. Brouns, Louis Reese
bioRxiv 471482; doi: https://doi.org/10.1101/471482

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