CRISPR-based DNA and RNA detection with liquid phase separation

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).

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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Contributors

Author: Franklin Luzia De Nobrega

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