Detection of nucleic acid biomarkers is on the forefront of biomolecular and diagnostic research, especially with the current COVID-19 pandemic, during which several new methods have been developed for the detection of SARS-CoV-2 viral RNA. Now, researchers from the Delft University of Technology (Netherlands) and University of Southampton (UK) have developed a CRISPR-based method that provides a visual readout based on liquid-liquid phase separation.

CRISPR-Cas enzymes are used in gene editing and diagnostics because of their non-specific cleavage of DNA or RNA sequences upon binding to a specific target nucleic acid. In a new study, researchers combined CRISPR technology with a biophysical phenomenon called liquid-liquid phase separation (LLPS). Due to their molecular and chemical properties, macromolecules can phase separate into a polymer-rich (coacervate) phase and a polymer-depleted (solvent) phase. For nucleic acids, this is known to be dependent on the length of the DNA or RNA molecules, with longer fragments readily phase separating compared with shorter fragments (see Figure 1).

In this study, researchers tested different lengths of DNA and RNA to find out the optimal length for phase separation. They used a 60-nucleotide poly-T DNA as “collateral polymer” and poly-L-lysine as oppositely charged polyelectrolytes to cause LLPS, resulting in a turbid solution. CRISPR enzymes were added to this solution, followed by a target nucleic acid sample. In the presence of the target, the CRISPR Cas12a enzyme activates and cleaves the poly-T DNA, reducing the phase separation and resulting in a transparent solution. This visual change in solution turbidity indicates the presence of the target DNA. The group modified this system to detect RNA by using a poly-U RNA sequence as the collateral polymer and tetravalent polycation spermine as the oppositely charged molecule to cause LLPS. For the readout, they used Cas13a enzyme that can degrade RNA (Figure 2).

As a biosensing assay, this method has a detection limit in the sub-micromolar concentration for single stranded DNA targets and in the nanomolar range for RNA targets. This method provides direct detection without any amplification steps, but has the potential to be combined with PCR or Recombinase Polymerase Amplification (RPA) to obtain higher sensitivity. One key aspect is that the solution conditions have to be optimized for each case to enable phase separation for specific targets. In the context of alternate nucleic acid detection methods, this technique provides, label-free, direct detection of nucleic acids, and could be a valuable addition to the suite of novel technologies that are being developed for nucleic acid diagnostics.

Read more about their study in “CRISPR-based DNA and RNA detection with liquid-liquid phase separation,” published in Volume 120, Issue 7 of Biophysical Journal.