Detection of tomato brown rugose fruit virus through CRISPR-Cas12a and CRISPR-Cas9 systems.

Tomato brown rugose fruit virus (ToBRFV) is a single-stranded positive-sense RNA virus that targets tomato and pepper plants and is causing significant damage to crops in some regions of the world. ToBRFV is a highly contagious virus that is stable and rapidly spreads by mechanical methods and seeds. As a result, it may spread both locally and over long distances, and it is now recognized as a pandemic in plants.

Arabidopsis thaliana Iron Superoxide Dismutase FeSOD1 Protects ARGONAUTE 1 in a Copper-Dependent Manner.

Copper (Cu) deficiency compromises plant growth and limits crop productivity. Plants respond to Cu scarcity by activating the expression of several microRNAs, known as Cu-miRNAs, which degrade mRNAs from various cuproproteins to conserve Cu. Cu-miRNAs, like most plant miRNAs, associate with ARGONAUTE 1 (AGO1), the primary effector protein of miRNA-mediated gene silencing pathways, whose function is typically modulated by interacting proteins acting as cofactors.

Digitizing the Blue Light-Activated T7 RNA Polymerase System with a -Controlled Riboregulator.

Optogenetic systems offer precise control over gene expression, but leaky activity in the dark limits their dynamic range and, consequently, their applicability. Here, we enhanced an optogenetic system based on a split T7 RNA polymerase fused to blue-light-inducible Magnets by incorporating a -controlled riboregulatory module. This module exploits the photosensitivity of anhydrotetracycline and the designability of synthetic small RNAs to digitize light-controlled gene expression, implementing a repressive action over the translation of a polymerase fragment gene that is relieved with blue light.

Virus Detection by CRISPR-Cas9-Mediated Strand Displacement in a Lateral Flow Assay.

In public health emergencies or in resource-constrained settings, laboratory-based diagnostic methods, such as RT-qPCR, need to be complemented with accurate, rapid, and accessible approaches to increase testing capacity, as this will translate into better outcomes in disease prevention and management. Here, we develop an original nucleic acid detection platform by leveraging CRISPR-Cas9 and lateral flow immunochromatography technologies. In combination with an isothermal amplification that runs with a biotinylated primer, the system exploits the interaction between the CRISPR-Cas9 R-loop formed upon targeting a specific nucleic acid and a fluorescein-labeled probe to generate a visual readout on a lateral flow device.

Regulatory Effects of RNA-Protein Interactions Revealed by Reporter Assays of Bacteria Grown on Solid Media.

Reporter systems are widely used to study biomolecular interactions and processes in vivo, representing one of the basic tools used to characterize synthetic regulatory circuits. Here, we developed a method that enables the monitoring of RNA-protein interactions through a reporter system in bacteria with high temporal resolution. For this, we used a Real-Time Protein Expression Assay (RT-PEA) technology for real-time monitoring of a fluorescent reporter protein, while having bacteria growing on solid media.

Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli.

RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli.

Rapid and Accurate Detection of the SARS-CoV-2 Omicron Variant with a CRISPR-Cas12a Reaction in the RT-qPCR Pot.

Gene sequencing in back of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the current approach for discriminating infections produced by different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the clinic. However, sequencing is often a time-consuming step, which hinders the deployment of a very fast response during a pandemic. Here, we propose to run a CRISPR-Cas12a reaction after completing the RT-qPCR and in the very same pot to detect with high specificity genetic marks characterizing variants of concern.

SARS-CoV-2 remodels the landscape of small non-coding RNAs with infection time and symptom severity.

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 has significantly impacted global health, stressing the necessity of basic understanding of the host response to this viral infection. In this study, we investigated how SARS-CoV-2 remodels the landscape of small non-coding RNAs (sncRNA) from a large collection of nasopharyngeal swab samples taken at various time points from patients with distinct symptom severity. High-throughput RNA sequencing analysis revealed a global alteration of the sncRNA landscape, with abundance peaks related to species of 21-23 and 32-33 nucleotides.

On the ever-growing functional versatility of the CRISPR-Cas13 system.

CRISPR-Cas systems evolved in prokaryotes to implement a powerful antiviral immune response as a result of sequence-specific targeting by ribonucleoproteins. One of such systems consists of an RNA-guided RNA endonuclease, known as CRISPR-Cas13. In very recent years, this system is being repurposed in different ways in order to decipher and engineer gene expression programmes.

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria.

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs.

Plant virus-derived nanoparticles decorated with genetically encoded SARS-CoV-2 nanobodies display enhanced neutralizing activity.

Viral nanoparticles (VNPs) are a new class of virus-based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self-assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant.

A variant-dependent molecular clock with anomalous diffusion models SARS-CoV-2 evolution in humans.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in humans has been monitored at an unprecedented level due to the public health crisis, yet the stochastic dynamics underlying such a process is dubious. Here, considering the number of acquired mutations as the displacement of the viral particle from the origin, we performed biostatistical analyses from numerous whole genome sequences on the basis of a time-dependent probabilistic mathematical model. We showed that a model with a constant variant-dependent evolution rate and nonlinear mutational variance with time (i.

Multiplexable and Biocomputational Virus Detection by CRISPR-Cas9-Mediated Strand Displacement.

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal.

Programmable regulation of translation by harnessing the CRISPR-Cas13 system.

The ability to control protein expression at both the transcriptional and post-transcriptional levels is instrumental for the cell to integrate multiple molecular signals and then reach high operational sophistication. Although challenging, fully artificial regulations at different levels are required for boosting systems and synthetic biology. Here, we report the development of a novel framework to regulate translation by repurposing the CRISPR-Cas13 immune system, which uses an RNA-guided ribonuclease.

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes.

Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions.

Metal-organic frameworks in pursuit of size: the development of macroscopic single crystals.

Metal-organic frameworks are versatile structures with many different applications, from industry to the clinic. Despite multiple synthesis approaches being able to coordinate metals and organic ligands, some common strategies can be employed when the size of the final product is a target. Here, we review the most relevant examples of synthesized crystals of very large size (, macroscopic, in the scale of mm), emphasizing the critical parameters that impact the final crystal size and quality.

Gene regulation by a protein translation factor at the single-cell level.

Gene expression is inherently stochastic and pervasively regulated. While substantial work combining theory and experiments has been carried out to study how noise propagates through transcriptional regulations, the stochastic behavior of genes regulated at the level of translation is poorly understood. Here, we engineered a synthetic genetic system in which a target gene is down-regulated by a protein translation factor, which in turn is regulated transcriptionally.

CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation.

The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the potential of increasing its transmission and the induced disease. Here, we applied the CRISPR-Cas12a system to detect, without the need of sequencing, SARS-CoV-2 genomes harboring the E484K mutation, first identified in the Beta variant and catalogued as an escape mutation. The E484K mutation creates a canonical protospacer adjacent motif for Cas12a recognition in the resulting DNA amplicon, which was exploited to obtain a differential readout.

Molecular signatures of silencing suppression degeneracy from a complex RNA virus.

As genomic architectures become more complex, they begin to accumulate degenerate and redundant elements. However, analyses of the molecular mechanisms underlying these genetic architecture features remain scarce, especially in compact but sufficiently complex genomes. In the present study, we followed a proteomic approach together with a computational network analysis to reveal molecular signatures of protein function degeneracy from a plant virus (as virus-host protein-protein interactions).

Nonequilibrium thermodynamics of the RNA-RNA interaction underlying a genetic transposition program.

Thermodynamic descriptions are powerful tools to formally study complex gene expression programs evolved in living cells on the basis of macromolecular interactions. While transcriptional regulations are often modeled in the equilibrium, other interactions that occur in the cell follow a more complex pattern. Here, we adopt a nonequilibrium thermodynamic scheme to explain the RNA-RNA interaction underlying IS10 transposition.

CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA.

DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA.

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus.

A complex network of cellular receptors, RNA targeting pathways, and small-molecule signaling provides robust plant immunity and tolerance to viruses. To maximize their fitness, viruses must evolve control mechanisms to balance host immune evasion and plant-damaging effects. The genus Potyvirus comprises plant viruses characterized by RNA genomes that encode large polyproteins led by the P1 protease.

Engineering a Circular Riboregulator in .

RNAs of different shapes and sizes, natural or synthetic, can regulate gene expression in prokaryotes and eukaryotes. Circular RNAs have recently appeared to be more widespread than previously thought, but their role in prokaryotes remains elusive. Here, by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme, we created a small noncoding RNA that self-splices to produce a circular riboregulator in .

Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time.

Synthetic biology aims at engineering gene regulatory circuits to end with cells (re)programmed on purpose to implement novel functions or discover natural behaviors. However, one overlooked question is whether the resulting circuits perform as intended in variety of environments or with time. Here, we considered a recently engineered genetic system that allows programming the cell to work as a minimal computer (arithmetic logic unit) in order to analyze its operability regime.