Computational protein design with evolutionary-based and physics-inspired modeling: current and future synergies

Computational protein design facilitates discovery of novel proteins with prescribed structure and functionality. Exciting designs were recently reported using novel data-driven methodologies that can be roughly divided into two categories: evolutionary-based and physics-inspired approaches. The former infer characteristic sequence features shared by sets of evolutionary-related proteins, such as conserved or coevolving positions, and recombine them to generate candidates with similar structure and function. The latter estimate key biochemical properties such as structure free energy, conformational entropy or binding affinities using machine learning surrogates, and optimize them to yield improved designs. Here, we review recent progress along both tracks, discuss their strengths and weaknesses, and highlight opportunities for synergistic approaches

Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.National Institutes of Health (U.S.) (Pioneer Award DP1MH100706)National Institutes of Health (U.S.) (Transformative Research Award)W. M. Keck FoundationMcKnight FoundationBill & Melinda Gates FoundationDamon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramEsther A. & Joseph Klingenstein Fund, Inc.Simons Foundatio

The synthetic integron: an in vivo genetic shuffling device

As the field of synthetic biology expands, strategies and tools for the rapid construction of new biochemical pathways will become increasingly valuable. Purely rational design of complex biological pathways is inherently limited by the current state of our knowledge. Selection of optimal arrangements of genetic elements from randomized libraries may well be a useful approach for successful engineering. Here, we propose the construction and optimization of metabolic pathways using the inherent gene shuffling activity of a natural bacterial site-specific recombination system, the integron. As a proof of principle, we constructed and optimized a functional tryptophan biosynthetic operon in Escherichia coli. The trpA-E genes along with ‘regulatory’ elements were delivered as individual recombination cassettes in a synthetic integron platform. Integrase-mediated recombination generated thousands of genetic combinations overnight. We were able to isolate a large number of arrangements displaying varying fitness and tryptophan production capacities. Several assemblages required as many as six recombination events and produced as much as 11-fold more tryptophan than the natural gene order in the same context

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens

Frequency-dependent selection predicts patterns of radiations and biodiversity

Most empirical studies support a decline in speciation rates through time, although evidence for constant speciation rates also exists. Declining rates have been explained by invoking niche-filling processes, whereas constant rates have been attributed to non-adaptive processes such as sexual selection, mutation, and dispersal. Trends in speciation rate and the processes underlying it remain unclear, representing a critical information gap in understanding patterns of global diversity. Here we show that the speciation rate is driven by frequency dependent selection. We used a frequency-dependent and DNA sequence-based model of populations and genetic-distance-based speciation, in the absence of adaptation to ecological niches. We tested the frequency-dependent selection mechanism using cichlid fish and Darwin's finches, two classic model systems for which speciation rates and richness data exist. Using negative frequency dependent selection, our model both predicts the declining speciation rate found in cichlid fish and explains their species richness. For groups like the Darwin's finches, in which speciation rates are constant and diversity is lower, the speciation rate is better explained by a model without frequency-dependent selection. Our analysis shows that differences in diversity are driven by larger incipient species abundance (and consequent lower extinction rates) with frequency-dependent selection. These results demonstrate that mutations, genetic-distance-based speciation, sexual and frequency-dependent selection are sufficient not only for promoting rapid proliferation of new species, but also for maintaining the high diversity observed in natural systems

Does Sex Speed Up Evolutionary Rate and Increase Biodiversity?

Most empirical and theoretical studies have shown that sex increases the rate of evolution, although evidence of sex constraining genomic and epigenetic variation and slowing down evolution also exists. Faster rates with sex have been attributed to new gene combinations, removal of deleterious mutations, and adaptation to heterogeneous environments. Slower rates with sex have been attributed to removal of major genetic rearrangements, the cost of finding a mate, vulnerability to predation, and exposure to sexually transmitted diseases. Whether sex speeds or slows evolution, the connection between reproductive mode, the evolutionary rate, and species diversity remains largely unexplored. Here we present a spatially explicit model of ecological and evolutionary dynamics based on DNA sequence change to study the connection between mutation, speciation, and the resulting biodiversity in sexual and asexual populations. We show that faster speciation can decrease the abundance of newly formed species and thus decrease long-term biodiversity. In this way, sex can reduce diversity relative to asexual populations, because it leads to a higher rate of production of new species, but with lower abundances. Our results show that reproductive mode and the mechanisms underlying it can alter the link between mutation, evolutionary rate, speciation and biodiversity and we suggest that a high rate of evolution may not be required to yield high biodiversity

Etude du mécanisme de recombinaison des intégrons, et leur utilisation comme générateur de combinaisons génétiques à des fins biotechnologiques

Integrons are bacterial recombination systems that play a major in the spread of antibiotic resistance genes. These genetic platforms are able to capture gene cassettes and reorganize them to find adaptive solution to changing environments. The mechanism of integron recombination is unusual. In contrast to the other site-specific recombination systems of the same family (catalysed by a tyrosine recombinase), the recombination site of gene cassettes is recognized as folded single-stranded DNA. Half of my thesis work was dedicated to understanding how and when these recombination sites are able to go from the stable double-helix form of DNA to a conformation allowing recombination. The other half consisted in using the remarkable recombination capacities of integrons to develop a genetic shuffling device that can be used for the directed evolution of synthetic gene networks and metabolic pathways. Such device can indeed be of great use to genetic engineers trying to assemble new genetic pathways implementing functions of interest.Les integrons sont des systèmes de recombinaison génétique bactériens qui jouent un rôle majeur dans la dissémination des gènes de résistances aux antibiotiques. Ces plateformes génétiques sont capables de capturer des cassettes de gène et de les réorganiser afin de trouver des solutions adaptatives à un environnement changeant. Le mécanisme de recombinaison des intégrons est original. Contrairement aux autres systèmes de recombinaison spécifique de site de la même famille (catalysés par les recombinases à tyrosine), le site de recombinaison associé aux cassettes est reconnu sous forme d'ADN simple brin replié. Une large part de mon travail de thèse a été dédiée à la compréhension des mécanismes qui permettent à ces sites de recombinaison de passer de la forme stable qu'est la double hélice d'ADN à la conformation leur permettant d'être reconnu par la recombinase des intégrons. L'autre partie de ma thèse a consisté au développement d'un outil génétique utilisant les remarquables propriétés de recombinaison des intégrons. Ce nouvel outil, nommé « intégron synthétique » permet de générer un grand nombre de combinaisons de séquences hétérologues in vivo. Il pourrait être d'une grande utilité aux ingénieurs tentant d'assembler des réseaux et voies génétiques d'intérêt, par évolution dirigée

CRISPR Tools To Control Gene Expression in Bacteria

International audienceCRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions