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

[EN] 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. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.We thank V. Aragones (IBMCP) for her technical assistance on PAGE. The work was supported by the Spanish Ministry of Economy and Competitiveness grants BFU2015-66894-P (to GR) and BIO2017-83184-R (to JAD) and by the Spanish Ministry of Science, Innovation, and Universities grant PGC2018-101410-B-I00 (to GR); grants cofinanced by the European Regional Development Fund.Montagud-Martínez, R.; Heras-Hernández, M.; Goiriz, L.; Rodrigo Tarrega, G.; Daròs, J. (2021). CRISPR-Mediated Strand Displacement Logic Circuits with Toehold-Free DNA. ACS Synthetic Biology. 10(5):950-956. https://doi.org/10.1021/acssynbio.0c0064995095610

Fitness Trade-Offs Determine the Role of the Molecular Chaperonin GroEL in Buffering Mutations

Molecular chaperones fold many proteins and their mutated versions in a cell and can sometimes buffer the phenotypic effect of mutations that affect protein folding. Unanswered questions about this buffering include the nature of its mechanism, its influence on the genetic variation of a population, the fitness trade-offs constraining this mechanism, and its role in expediting evolution. Answering these questions is fundamental to understand the contribution of buffering to increase genetic variation and ecological diversification. Here, we performed experimental evolution, genome resequencing, and computational analyses to determine the trade-offs and evolutionary trajectories of Escherichia coli expressing high levels of the essential chaperonin GroEL. GroEL is abundantly present in bacteria, particularly in bacteria with large loads of deleterious mutations, suggesting its role in mutational buffering. We show that groEL overexpression is costly to large populations evolving in the laboratory, leading to groE expression decline within 66 generations. In contrast, populations evolving under the strong genetic drift characteristic of endosymbiotic bacteria avoid extinction or can be rescued in the presence of abundant GroEL. Genomes resequenced from cells evolved under strong genetic drift exhibited significantly higher tolerance to deleterious mutations at high GroEL levels than at native levels, revealing that GroEL is buffering mutations in these cells. GroEL buffered mutations in a highly diverse set of proteins that interact with the environment, including substrate and ion membrane transporters, hinting at its role in ecological diversification. Our results reveal the fitness trade-offs of mutational buffering and how genetic variation is maintained in population

The Molecular Chaperone DnaK Is a Source of Mutational Robustness

[EN] Molecular chaperones, also known as heat-shock proteins, refold misfolded proteins and help other proteins reach their native conformation. Thanks to these abilities, some chaperones, such as the Hsp90 protein or the chaperonin GroEL, can buffer the deleterious phenotypic effects of mutations that alter protein structure and function. Hsp70 chaperones use a chaperoning mechanism different from that of Hsp90 and GroEL, and it is not known whether they can also buffer mutations. Here, we show that they can. To this end, we performed a mutation accumulation experiment in Escherichia coli, followed by whole-genome resequencing. Overexpression of the Hsp70 chaperone DnaK helps cells cope with mutational load and completely avoid the extinctions we observe in lineages evolving without chaperone overproduction. Additionally, our sequence data show that DnaK overexpression increases mutational robustness, the tolerance of its clients to nonsynonymous nucleotide substitutions. We also show that this elevated mutational buffering translates into differences in evolutionary rates on intermediate and long evolutionary time scales. Specifically, we studied the evolutionary rates of DnaK clients using the genomes of E. coli, Salmonella enterica, and 83 other gamma-proteobacteria. We find that clients that interact strongly with DnaK evolve faster than weakly interacting clients. Our results imply that all three major chaperone classes can buffer mutations and affect protein evolution. They illustrate how an individual protein like a chaperone can have a disproportionate effect on the evolution of a proteome.The authors thank Xiaoshu Chen and Jianzhi Zhang for kindly providing us with the gene expression data. This work was supported by the Forschungskredit program of the University of Zurich (grant FK-14-076 to J.A.), the Swiss National Science Foundation (grant 31003A_146137 to A.W.), the University Priority Research Program in Evolutionary Biology at the University of Zurich (to A.W.), the Science Foundation Ireland (grant 12/IP/1673 to M.A.F.), and the Spanish Ministerio de Economia y Competitividad (grant BFU2012-36346 to M.A.F.). We posted an earlier version of this paper in bioRxiv (doi: http://dx.doi.org/10.1101/040600) on 22 February 2016.Aguilar-Rodríguez, J.; Sabater-Munoz, B.; Montagud-Martinez, R.; Berlanga, V.; Alvarez-Ponce, D.; Wagner, A.; Fares Riaño, MA. (2016). The Molecular Chaperone DnaK Is a Source of Mutational Robustness. Genome Biology and Evolution. 8(9):2979-2991. https://doi.org/10.1093/gbe/evw176S297929918

Study of Spatiotemporal Responses of Bacterial Cells

[ES] La biotecnología moderna se basa en la aplicación de una mezcla de herramientas experimentales y computacionales para llevar a cabo de forma dirigida la ingeniería genética. El objetivo es obtener células (re)programadas que implementen nuevas funciones o que sirvan como herramientas para el estudio de sistemas biológicos. En este contexto, el uso de bacterias en biotecnología está muy extendido. Sin embargo, la implementación de circuitos genéticos para el aprovechamiento de estos seres vivos puede verse limitada por procesos biológicos naturales; es decir, los circuitos diseñados (o naturales) pueden verse afectados por el transcurso del tiempo o por cambios en el entorno en el que crecen las bacterias. En esta tesis, nos propusimos seguir un enfoque integrador para estudiar cómo las bacterias responden en el tiempo y el espacio a los cambios genéticos y ambientales, que pueden afectar la funcionalidad de los circuitos de interés biotecnológico. Usamos Escherichia coli como organismo modelo, explotando una variedad de herramientas experimentales para trabajar con él. En primer lugar, estudiamos cómo los cambios ambientales y genéticos afectan la funcionalidad de un circuito genético sintético que implementa un comportamiento lógico sofisticado. Descubrimos que hay amplios rangos de concentración de entrada que el sistema puede procesar correctamente, que el circuito diseñado es bastante sensible a los efectos de la temperatura, que la expresión de pequeños ARN heterólogos es costosa para la célula y que una reorganización genética adecuada del sistema para reducir la cantidad de ADN heterólogo en la célula puede mejorar su estabilidad evolutiva. En segundo lugar, estudiamos el crecimiento bacteriano en entornos en los que existen materiales nanoestructurados. Descubrimos que las poblaciones bacterianas se pueden controlar en gran medida mediante el uso de marcos organometálicos, ya que estos materiales nanoestructurados pueden descomponerse lentamente en medios biológicos liberando agentes antimicrobianos (metales y compuestos orgánicos, incluidos los antibióticos). Analizamos la respuesta bacteriana espaciotemporal siguiendo un enfoque experimental y teórico combinado en un entorno tan complejo y desafiante en medios líquidos y sólidos. Además de las variaciones en el rendimiento debido a cambios ambientales, también se debe considerar que esos circuitos genéticos evolucionarán con el tiempo debido a la acumulación estocástica de mutaciones. Estas mutaciones pueden dar lugar a cambios en la funcionalidad de los circuitos reguladores. Por tanto, en tercer lugar, realizamos un experimento de evolución a largo plazo para estudiar la contribución de un sistema de chaperonas de proteínas en la modulación de la estabilidad evolutiva. En los últimos años, se ha demostrado que los sistemas de chaperonas, como GroES/EL, pueden amortiguar o purgar mutaciones. Realizamos la secuenciación del genoma completo en diferentes líneas con diferentes niveles de expresión de GroEL y también medimos la tasa de crecimiento de las células al principio y al final del experimento evolutivo. Sin embargo, nuestros resultados no fueron concluyentes, por lo que se necesita más investigación para comprender completamente el papel de GroES/EL en la evolución y evaluar su utilidad potencial en biotecnología. En conjunto, esta tesis intenta avanzar en nuestro conocimiento sobre cómo las bacterias, y E. coli en particular, se comportan como se espera cuando el entorno se altera, la fisiología cambia y pasa mucho tiempo, para posibles aplicaciones industriales o (pre)clínicas.[CA] La biotecnologia moderna es basa en l'aplicació d'una mescla d'eines experimentals i computacionals per a realitzar de forma dirigida l'enginyeria genètica. L'objectiu és obtindre cèl·lules (re)programades que implementen noves funcions o que servisquen com a eines per a l'estudi de sistemes biològics. En aquest context, l'ús de bacteris en biotecnologia està molt estés. No obstant això, la implementació de circuits genètics per a l'aprofitament d'aquests éssers vius pot veure's limitada per processos biològics naturals; és a dir, els circuits dissenyats (o naturals) poden veure's afectats pel transcurs del temps o per canvis en l'entorn en el qual creixen els bacteris. En aquesta tesi, ens vam proposar seguir un enfocament integrador per a estudiar com els bacteris responen en el temps i l'espai als canvis genètics i ambientals, que poden afectar la funcionalitat dels circuits d'interés biotecnològic. Usem Escherichia coli com a organisme model, explotant una varietat d'eines experimentals per a treballar amb ell. En primer lloc, estudiem com els canvis ambientals i genètics afecten la funcionalitat d'un circuit genètic sintètic que implementa un comportament lògic sofisticat. Descobrim que hi ha amplis rangs de concentració d'entrada que el sistema pot processar correctament, que el circuit dissenyat és bastant sensible a l'efecte de la temperatura, que l'expressió de xicotets ARN heteròlegs és costosa per a la cèl·lula i que una reorganització genètica adequada del sistema per a reduir la quantitat d'ADN heteròleg en la cèl·lula pot millorar la seua estabilitat evolutiva. En segon lloc, estudiem el creixement bacterià en entorns en els quals existeixen materials nanoestructurats. Descobrim que les poblacions bacterianes es poden controlar en gran manera mitjançant l'ús de marcs organometàlics, ja que aquests materials nanoestructurats poden descompondre's lentament en medis biològics alliberant agents antimicrobians (metalls i compostos orgànics, inclosos els antibiòtics). Analitzem la resposta bacteriana espai-temporal seguint un enfocament experimental i teòric integrador en un entorn tan complex i desafiador en mitjans líquids i sòlids. A més de les variacions en el rendiment degut a canvis ambientals, també s'ha de considerar que aqueixos circuits genètics evolucionaran amb el temps degut a l'acumulació estocàstica de mutacions. Aquestes mutacions poden donar lloc a canvis en la funcionalitat dels circuits reguladors. Per tant, en tercer lloc, realitzem un experiment d'evolució a llarg termini per a estudiar la contribució d'un sistema de chaperones de proteïnes en la modulació de l'estabilitat evolutiva. En els últims anys, s'ha demostrat que els sistemes de chaperones, com GroES/EL, poden esmorteir o purgar mutacions. Realitzem la seqüenciació del genoma complet en diferents línies amb diferents nivells d'expressió de GroEL i també mesurem la taxa de creixement de les cèl·lules al principi i al final de l'experiment evolutiu. No obstant això, els nostres resultats no van ser concloents, per la qual cosa es necessita més investigació per a comprendre completament el paper de GroES/L en l'evolució i avaluar la seua utilitat potencial en biotecnologia. En conjunt, aquesta tesi intenta avançar en el nostre coneixement sobre com els bacteris, i E. coli en particular, es comporten com s'espera quan l'entorn s'altera, la fisiologia canvia i passa molt temps, per a possibles aplicacions industrials o (pre)clíniques.[EN] Modern biotechnology is based on applying a mix of experimental and computational tools to perform in a directed way genetic engineering. The aim is to obtain (re)programmed cells that implement new functions or that serve as tools for the study of biological systems. In this context, the use of bacteria in biotechnology is widespread. However, the implementation of genetic circuits for the use of these living beings may be limited due to natural biological processes; that is, the engineered (or natural) circuits may be affected by the course of time or by changes in the environment in which bacteria grow. In this thesis, we proposed to follow an integrative approach to study how bacteria respond in time and space to genetic and environmental changes, which may affect the functionality of the circuits of biotechnological interest. We used Escherichia coli as a model organism, exploiting a variety of experimental tools to work with it. Firstly, we studied how environmental and genetic changes affect the functionality of a synthetic genetic circuit that implements a sophisticated logic behavior. We found that there are wide input concentration ranges that the system can correctly process, that the engineered circuitry is quite sensitive to temperature effects, that the expression of heterologous small RNAs is costly for the cell, and that a proper genetic reorganization of the system to reduce the amount of heterologous DNA in the cell can improve its evolutionary stability. Secondly, we studied of bacterial growth in environments in which there are nanostructured materials. We found that bacterial populations can be greatly controlled through the use of metal-organic frameworks, as these nanostructured materials can slowly decompose in biological media releasing antimicrobials (metals and organic compounds, including antibiotics). We analyzed the spatiotemporal bacterial response following a combined experimental and theoretical approach in a such a complex and challenging environment in both liquid and solid media. In addition to variations in performance due to environmental changes, it must also be considered that those gene circuits will evolve over time due to the stochastic accumulation of mutations. These mutations can lead to changes in the functionality of the regulatory circuits. Then thirdly, we performed an experiment of long-term evolution to study the contribution of a protein chaperone system in modulating evolutionary stability. In recent years, it has been shown that chaperone systems, such as GroES/EL, can buffer or purge mutations. We performed whole-genome sequencing over different lines with varying expression levels of GroEL, and also measured the growth rate of the cells at the beginning and the end of the evolutionary experiment. However, our results were not conclusive, so further research is needed to fully understand the role of GroES/EL in evolution and to assess its potential utility in biotechnology. Taken together, this thesis tries to advance our knowledge on how bacteria, and E. coli in particular, behave as expected when the environment is perturbed, the physiology changes, and long time passes, for potential industrial or (pre)clinical applications.Montagud Martínez, R. (2023). Study of Spatiotemporal Responses of Bacterial Cells [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/19303

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. We analyzed a series of fecal samples from hospitalized patients in Valencia (Spain), finding one infection with SARS-CoV-2 harboring the E484K mutation, which was then confirmed by sequencing. Overall, these results suggest that CRISPR diagnostics can be a useful tool in epidemiology to monitor the spread of escape mutationsThis work was supported by the Fondo Supera COVID-19 from CRUE and Banco Santander (Grant COVCRISPIS, BOE-A-2020-7995, to GR), the CSIC PTI Salud Global (Grant SGL2021-03-040, to GR, and Grants 202020E292 and SGL2103034, to PDC) through the European Union−NextGeneration EU, the Fondo COVID-19 from the Instituto de Salud Carlos III (Grant COV20/00210, to PDC), and the Generalitat Valenciana (Grant SEJI/2020/ 011, to GR). PDC was supported by the Ramón y Cajal program (RYC2019-028015-I) and RMC by a predoctoral fellowship (PRE2019-088531), both from the Spanish Ministerio de Ciencia e Innovación.Peer reviewe

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. By exploiting a cell-free expression system for prototyping gene regulatory structures, our results demonstrate that CRISPR-dCas13a ribonucleoproteins (d means catalytically dead) can be programmed to repress or activate translation initiation. The performance assessment of the engineered systems also revealed guide RNA design principles. Moreover, we show that the system can work in vivo. This development complements the ability to regulate transcription with other CRISPR-Cas systems and offers potential applications.This work was supported by the grants PGC2018-101410-B-I00 from the Spanish Ministry of Science, Innovation, and Universities (co-financed by the European Regional Development Fund) and SEJI/2020/011 from the Regional Government of Valencia (to GR). RMC held a predoctoral fellowship (PRE2019-088531).Publication fees covered by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research.Peer reviewe

Bacterial population control with macroscopic HKUST crystals

Macroscopic HKUST crystals were shown to release significant amounts of copper in saline medium at a slow rate, which was exploited to control the growth of a bacterial population. This was achieved in both liquid and solid media, the latter illustrating the local effect of the crystals. In addition, these nanostructured crystals of observable size were loaded with chloramphenicol to exert a joint metal-antibiotic action, going beyond the traditional oligodynamic effect.This work is supported by the Spanish Ministry of Economy, Industry, and Competitiveness (CONSOLIDER CTQ2017-90852-REDC).Peer reviewe

Fitness and mutational rate is affected by loss of mutational buffering in highly bottlenecked E. coli populations

Resumen del trabajo presentado al XLII Congreso de la Sociedad Española de Genética, celebrado de forma virtual del 14 al 18 de junio de 2021.Peer reviewe

Genomic changes and the importance of the chaperonin GroEL in the evolution of highly bottlenecked bacterial populations

Resumen del póster presentado al Society for Molecular Biology and Evolution Meeting (SMBEv), celebrado de forma virtual del 3 al 8 de julio de 2021.Chaperones are involved in the folding of nascent client proteins, the prevention of polypeptides aggregation, the rescue of unfolded clients due to environmental stresses and act as an evolutionary driver due to their mutational buffering capacities. Major insect lineages have independently acquired bacterial species, mainly from Gamma-proteobacteria and Bacteroidetes class. These bacterial species could act as nutritional mutualistic factories, facultative mutualists that protect against biotic and abiotic stresses, or reproductive manipulators. Common trade among them is an increased level of genetic drift due to the small population size and the continuous population bottlenecking at each generation, processes that have shaped their genome, proteome, and morphology. Depending on the nature of the relationship, the degree of genome plasticity varies, i.e., obligate nutritional mutualistic symbionts have extremely small genomes lacking mobile elements, bacteriophages, and/or recombination machinery. Under these conditions, endosymbionts face high mutational pressures that may lead to extinction or symbiont replacement. How do they then survive for such a long evolutionary time, and why do they show genome stasis? Here we will focus on the genome changes suffered by these endosymbionts, by comparing them to their free-living relatives, and on the mutational robustness mechanisms, including the moonlighting chaperone GroEL that could explain their long prevalence from an evolutionary perspective by using experimental evolution of E. coli to simulate the effect of high groEL overexpression and strong genetic drift.Peer reviewe

Loss of GroEL mutational buffering affects fitness and mutational rate under highly bottlenecked population dynamics

Trabajo presentado al Meeting of the Society for Molecular Biology and Evolution (SMBE), celebrado en Manchester (UK) del 21 al 25 de julio de 2019.Chaperones are involved in the folding of nascent client proteins, in the prevention of unfolded polypeptides aggregation and in the rescue of unfolded ones due to environmental stresses. The pioneering works of Rutheford and Lindquist, and those of Fares¿ team, and Touriki and Tawfik, have highlighted the importance of chaperones in buffering mutational effects by allowing for the adaptive evolution of its client proteins. These adaptive leaps might explain how ancient symbiosis still persist even under a strong genetic drift regime. But, how would an adapted consortium deal with the loss of this key system? When the organism lacks this rubustness system (as happens in many Mycoplasma species), proteome evolution becomes independent of protein folding. But what happens when the organism proteome is depending on its chaperone folding capabilities, and this system fails?. Experimental evolution of Escherichia coliunder high-expression rate of GroEL is only possible when the system is subjected to strong genetic drift, as overexpression is significantly costly. Despite this limitation, the loss of GroEL overexpression increase the extinction rate, observing an equilibrium between GroEL level and fitness. By challenging E. coli to daily single-cell bottlenecks under high GroEL overexpression, we found that after a certain number of generations, a number of compensatory mutations arose in the system allowing to decrease the GroEL level while not effecting the fitness. How these two parameters, structural stability and functional innovation interact, still deserves further research.Peer reviewe