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

The ability to detect specific nucleic acid sequences allows for a wide range of applications such as the 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 indiscriminate nucleic acid cleavage, which has been exploited for sequence-specific molecular diagnostics of nucleic acids. Here, we present a label-free detection approach that uses a readout based on solution turbidity caused by liquid-liquid phase separation (LLPS). Our approach relies on the fact that the LLPS of oppositely charged polymers requires polymers to be longer than a critical length. This length dependence is predicted by the Voorn-Overbeek model, which we describe in detail and validate experimentally in mixtures of polynucleotides and polycations. We show that the turbidity resulting from LLPS can be used to detect the presence of specific nucleic acid sequences by employing the programmable CRISPR-nucleases Cas12a and Cas13a. Because LLPS of polynucleotides and polycations causes solutions to become turbid, the detection of specific nucleic acid sequences can be observed with the naked eye. We furthermore demonstrate that there is an optimal polynucleotide concentration for detection. Finally, we provide a theoretical prediction that hints towards possible improvements of an LLPS-based detection assay. The deployment of LLPS complements CRISPR-based molecular diagnostic applications and facilitates easy and low-cost nucleotide sequence detection.

Nonspherical Coacervate Shapes in an Enzyme-Driven Active System.

Coacervates are polymer-rich droplets that form through liquid-liquid phase separation in polymer solutions. Liquid-liquid phase separation and coacervation have recently been shown to play an important role in the organization of biological systems. Such systems are highly dynamic and under continuous influence of enzymatic and chemical processes. However, it is still unclear how enzymatic and chemical reactions affect the coacervation process. Here, we present and characterize a system of enzymatically active coacervates containing spermine, RNA, free nucleotides, and the template independent RNA (de)polymerase PNPase. We find that these RNA coacervates display transient nonspherical shapes, and we systematically study how PNPase concentration, UDP concentration, and temperature affect coacervate morphology. Furthermore, we show that PNPase localizes predominantly into the coacervate phase and that its depolymerization activity in high-phosphate buffer causes coacervate degradation. Our observations of nonspherical coacervate shapes may have broader implications for the relationship between (bio)chemical activity and coacervate biology.

Spatiotemporal control of coacervate formation within liposomes.

Liquid-liquid phase separation (LLPS), especially coacervation, plays a crucial role in cell biology, as it forms numerous membraneless organelles in cells. Coacervates play an indispensable role in regulating intracellular biochemistry, and their dysfunction is associated with several diseases. Understanding of the LLPS dynamics would greatly benefit from controlled in vitro assays that mimic cells. Here, we use a microfluidics-based methodology to form coacervates inside cell-sized (~10 µm) liposomes, allowing control over the dynamics. Protein-pore-mediated permeation of small molecules into liposomes triggers LLPS passively or via active mechanisms like enzymatic polymerization of nucleic acids. We demonstrate sequestration of proteins (FtsZ) and supramolecular assemblies (lipid vesicles), as well as the possibility to host metabolic reactions (ß-galactosidase activity) inside coacervates. This coacervate-in-liposome platform provides a versatile tool to understand intracellular phase behavior, and these hybrid systems will allow engineering complex pathways to reconstitute cellular functions and facilitate bottom-up creation of synthetic cells.

Mechanical Division of Cell-Sized Liposomes.

Liposomes, self-assembled vesicles with a lipid-bilayer boundary similar to cell membranes, are extensively used in both fundamental and applied sciences. Manipulation of their physical properties, such as growth and division, may significantly expand their use as model systems in cellular and synthetic biology. Several approaches have been explored to controllably divide liposomes, such as shape transformation through temperature cycling, incorporation of additional lipids, and the encapsulation of protein division machinery. However, so far, these methods lacked control, exhibited low efficiency, and yielded asymmetric division in terms of volume or lipid composition. Here, we present a microfluidics-based strategy to realize mechanical division of cell-sized (∼6 µm) liposomes. We use octanol-assisted liposome assembly (OLA) to produce liposomes on chip, which are subsequently flowed against the sharp edge of a wedge-shaped splitter. Upon encountering such a Y-shaped bifurcation, the liposomes are deformed and, remarkably, are able to divide into two stable daughter liposomes in just a few milliseconds. The probability of successful division is found to critically depend on the surface area-to-volume ratio of the mother liposome, which can be tuned through osmotic pressure, and to strongly correlate to the mother liposome size for given microchannel dimensions. The division process is highly symmetric (∼3% size variation between the daughter liposomes) and is accompanied by a low leakage. This mechanical division of liposomes may constitute a valuable step to establish a growth-division cycle of synthetic cells.

Tailoring the appearance: what will synthetic cells look like?

Recently, the bottom-up assembly of a synthetic cell has emerged as a daring novel approach that can be expected to have major impact in generating fundamental insight in the organization and function of actual biological cells, as well as in stimulating a broad range of applications from drug delivery systems to chemical nanofactories. A crucial feature of any such synthetic cell is the architectural scaffold that defines its identity, compartmentalizes its inner content, and serves as a protective and selective barrier against its environment. Here we review a variety of potential scaffolds for building a synthetic cell. We categorize them as membranous structures (liposomes, fatty acid vesicles, polymersomes), emulsions (droplets and colloidosomes), and membrane-less coacervates. We discuss recent advances for each of them, and explore their salient features as candidates for designing synthetic cells.