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CRISPR-based DNA and RNA detection with liquid phase separation

CRISPR-based DNA and RNA detection with liquid phase separation
CRISPR-based DNA and RNA detection with liquid phase separation
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.
Spoelstra, Willem Kasper
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Jacques, Jeroen M.
6ecd1449-cd3f-415a-880a-71885caf5002
Luzia De Nobrega, Franklin
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Haagsma, Anna C
676809c5-23b4-435e-ad76-adf4982c2d72
Dogterom, Marileen
4643455e-82dc-460e-8f31-4f49afe73aea
Idema, Timon
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Brouns, Stan J J
b9c93a6a-120b-476b-8394-048e41d8ae79
Reese, Louis
fa6c544c-9fb3-4db2-96d1-74a476b422c6
Spoelstra, Willem Kasper
4a019318-8498-4c59-9a25-76a9584abf9b
Jacques, Jeroen M.
6ecd1449-cd3f-415a-880a-71885caf5002
Luzia De Nobrega, Franklin
6532795d-88a4-4f05-9b26-6af5b8f21a0d
Haagsma, Anna C
676809c5-23b4-435e-ad76-adf4982c2d72
Dogterom, Marileen
4643455e-82dc-460e-8f31-4f49afe73aea
Idema, Timon
5fe63b43-d0a5-4fdc-b6b2-98b0c232731e
Brouns, Stan J J
b9c93a6a-120b-476b-8394-048e41d8ae79
Reese, Louis
fa6c544c-9fb3-4db2-96d1-74a476b422c6

Spoelstra, Willem Kasper, Jacques, Jeroen M., Luzia De Nobrega, Franklin, Haagsma, Anna C, Dogterom, Marileen, Idema, Timon, Brouns, Stan J J and Reese, Louis (2019) CRISPR-based DNA and RNA detection with liquid phase separation. bioRxiv. (doi:10.1101/471482).

Record type: Article

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.

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More information

Published date: 15 May 2019

Identifiers

Local EPrints ID: 443075
URI: http://eprints.soton.ac.uk/id/eprint/443075
PURE UUID: da045697-6bc0-4d82-aff9-a2fc68c35021
ORCID for Franklin Luzia De Nobrega: ORCID iD orcid.org/0000-0002-8238-1083

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Date deposited: 10 Aug 2020 16:30
Last modified: 17 Mar 2024 04:02

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Contributors

Author: Willem Kasper Spoelstra
Author: Jeroen M. Jacques
Author: Anna C Haagsma
Author: Marileen Dogterom
Author: Timon Idema
Author: Stan J J Brouns
Author: Louis Reese

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