RESUMEN
The adoption of CRISPR systems for the generation of synthetic transcription factors has greatly simplified the process for upregulating endogenous gene expression, with a plethora of applications in cell biology, bioproduction and cell reprogramming. The recently discovered CRISPR/Cas12a (Cas12a) systems offer extended potential, as Cas12a is capable of processing its own crRNA array, to provide multiple individual crRNAs for subsequent targeting from a single transcript. Here we show the application of dFnCas12a-VPR in mammalian cells, with the Francisella novicida Cas12a (FnCas12a) possessing a shorter PAM sequence than Acidaminococcus sp. (As) or Lachnospiraceae bacterium (Lb) variants, enabling denser targeting of genomic loci, while performing just as well or even better than the other variants. We observe that synergistic activation and multiplexing can be achieved using crRNA arrays but also show that crRNAs expressed towards the 5' of 6-crRNA arrays show evidence of enhanced activity. This not only represents a more flexible tool for transcriptional modulation but further expands our understanding of the design capabilities and limitations when considering longer crRNA arrays for multiplexed targeting.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Endodesoxirribonucleasas/metabolismo , Edición Génica/métodos , Células HEK293 , Humanos , Empalme de ProteínaRESUMEN
Fluorescence microscopy enables specific visualization of proteins in living cells and has played an important role in our understanding of the protein subcellular location and function. Some proteins, however, show altered localization or function when labeled using direct fusions to fluorescent proteins, making them difficult to study in live cells. Additionally, the resolution of fluorescence microscopy is limited to â¼200 nm, which is 2 orders of magnitude larger than the size of most proteins. To circumvent these challenges, we previously developed LIVE-PAINT, a live-cell super-resolution approach that takes advantage of short interacting peptides to transiently bind a fluorescent protein to the protein-of-interest. Here, we successfully use LIVE-PAINT to image yeast membrane proteins that do not tolerate the direct fusion of a fluorescent protein by using peptide tags as short as 5-residues. We also demonstrate that it is possible to resolve multiple proteins at the nanoscale concurrently using orthogonal peptide interaction pairs.
Asunto(s)
Péptidos , Proteínas , Diagnóstico por Imagen , Saccharomyces cerevisiae , Colorantes Fluorescentes/químicaRESUMEN
The structure and diversity of all open microbial communities are shaped by individual births, deaths, speciation and immigration events; the precise timings of these events are unknowable and unpredictable. This randomness is manifest as ecological drift in the population dynamics, the importance of which has been a source of debate for decades. There are theoretical reasons to suppose that drift would be imperceptible in large microbial communities, but this is at odds with circumstantial evidence that effects can be seen even in huge, complex communities. To resolve this dichotomy we need to observe dynamics in simple systems where key parameters, like migration, birth and death rates can be directly measured. We monitored the dynamics in the abundance of two genetically modified strains of Escherichia coli, with tuneable growth characteristics, that were mixed and continually fed into 10 identical chemostats. We demonstrated that the effects of demographic (non-environmental) stochasticity are very apparent in the dynamics. However, they do not conform to the most parsimonious and commonly applied mathematical models, where each stochastic event is independent. For these simple models to reproduce the observed dynamics we need to invoke an 'effective community size', which is smaller than the census community size.
Asunto(s)
Microbiota , Escherichia coli/genética , Modelos Teóricos , Dinámica PoblacionalRESUMEN
CRISPR-enabled deaminase base editing has become a powerful tool for precisely editing nucleotides on the chromosome. In this study DNA helicases, such as Escherichia coli DnaB, were fused to activation-induced cytidine deaminase (AID) to form enzyme complexes which randomly introduces edited bases throughout the chromosome. DnaB-AID was found to increase 2.5 × 103 fold relative to the mutagenesis frequency of wildtype. 97.9% of these edits were observed on the leading strand during DNA replication suggesting deamination to be highly coordinated with DNA replication. Using DnaB-AID, a 371.4% increase in ß-carotene production was obtained following four rounds of editing. In Saccharomyces cerevisiae Helicase-AID was constructed by fusing AID to one of the subunits of eukaryotic helicase Mcm2-7 complex, MCM5. Using MCM5-AID, the average editing efficiency of five strains was 2.1 ± 0.4 × 103 fold higher than the native genomic mutation rate. MCM5-AID was able to improve ß-carotene production of S. cerevisiae 4742crt by 75.4% following eight rounds of editing. The S. cerevisiae MCM5-AID technique is the first biological tool for generating and accumulating single base mutations in eukaryotic chromosomes. Since the helicase complex is highly conservative in all eukaryotes, Helicase-AID could be adapted for various applications and research in all eukaryotic cells.
Asunto(s)
ADN Helicasas , Saccharomyces cerevisiae , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN Helicasas/metabolismo , Genoma , Genómica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
A device that counts and records the number of events experienced by an individual cell could have many uses in experimental biology and biotechnology. Here, we report a DNA-based 'latch' that switches between two states upon each exposure to a repeated stimulus. The key component of the latch is a DNA segment whose orientation is inverted by the actions of ÏC31 integrase and its recombination directionality factor (RDF). Integrase expression is regulated by an external input, while RDF expression is controlled by the state of the latch, such that the orientation of the invertible segment switches efficiently each time the device receives an input pulse. Recombination occurs over a time scale of minutes after initiation of integrase expression. The latch requires a delay circuit, implemented with a transcriptional repressor expressed in only one state, to ensure that each input pulse results in only one inversion of the DNA segment. Development and optimization of the latch in living cells was driven by mathematical modelling of the recombination reactions and gene expression regulated by the switch. We discuss how N latches built with orthogonal site-specific recombination systems could be chained together to form a binary ripple counter that could count to 2N - 1.
Asunto(s)
ADN/genética , Integrasas/genética , Recombinación Genética , Proteínas Virales/química , Bacteriófagos/genética , ADN/química , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Integrasas/química , Serina/genética , Análisis de la Célula Individual , Proteínas Virales/genéticaRESUMEN
Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ÏC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ÏC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices.
Asunto(s)
Integrasas/fisiología , Empalme de Proteína/genética , Recombinación Genética , Trans-Empalme/genética , Secuencia de Aminoácidos , Clonación Molecular/métodos , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Exteínas/genética , Integrasas/metabolismo , Inteínas/genética , Organismos Modificados Genéticamente , Ingeniería de Proteínas , Serina/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
Selectively fluorinated compounds are found frequently in pharmaceutical and agrochemical products where currently 25-30 % of optimised compounds emerge from development containing at least one fluorine atom. There are many methods for the site-specific introduction of fluorine, but all are chemical and they often use environmentally challenging reagents. Biochemical processes for C-F bond formation are attractive, but they are extremely rare. In this work, the fluorinase enzyme, originally identified from the actinomycete bacterium Streptomyces cattleya, is engineered into Escherichia coli in such a manner that the organism is able to produce 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-l-methionine (SAM) and fluoride in live E.â coli cells. Success required the introduction of a SAM transporter and deletion of the endogenous fluoride efflux capacity in order to generate an E.â coli host that has the potential for future engineering of more elaborate fluorometabolites.
Asunto(s)
Flúor/metabolismo , Ingeniería Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Flúor/química , Halogenación , Isomerismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , S-Adenosilmetionina/metabolismo , Streptomyces/enzimologíaRESUMEN
Yeast strains have been used extensively as robust microbial cell factories for the production of bulk and fine chemicals, including biofuels (bioethanol), complex pharmaceuticals (antimalarial drug artemisinin and opioid pain killers), flavours, and fragrances (vanillin, nootkatone, and resveratrol). In many cases, it is of benefit to suppress or modify ergosterol biosynthesis during strain engineering, for example, to increase thermotolerance or to increase metabolic flux through an alternate pathway. However, the impact of modifying ergosterol biosynthesis on engineered strains is discussed sparsely in literature, and little attention has been paid to the implications of these modifications on the general health and well-being of yeast. Importantly, yeast with modified sterol content exhibit a wide range of phenotypes, including altered organization and dynamics of plasma membrane, altered susceptibility to chemical treatment, increased tolerance to high temperatures, and reduced tolerance to other stresses such as high ethanol, salt, and solute concentrations. Here, we review the wide-ranging phenotypes of viable Saccharomyces cerevisiae strains with altered sterol content and discuss the implications of these for yeast as microbial cell factories.
Asunto(s)
Ergosterol/biosíntesis , Ingeniería Metabólica , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biocombustibles , Fermentación , Fenotipo , Esteroles/análisisRESUMEN
The CRISPR-Cas9 system has become increasingly popular for genome engineering across all fields of biological research, including in the Gram-positive model organism Bacillus subtilis. A major drawback for the commercial use of Cas9 is the IP landscape requiring a license for its use, as well as reach-through royalties on the final product. Recently an alternative CRISPR nuclease, free to use for industrial R&D, MAD7 was released by Inscripta (CO). Here we report the first use of MAD7 for gene editing in B. subtilis, in which editing rates of 93% and 100% were established. Additionally, we engineer the first reported catalytically inactive MAD7 (dMAD7) variant (D877A, E962A, and D1213A) and demonstrate its utility for CRISPR interference (CRISPRi) at up to 71.3% reduction of expression at single and multiplexed target sites within B. subtilis. We also confirm the CRISPR-based editing mode of action in B. subtilis providing evidence that the nuclease-mediated DNA double-strand break acts as a counterselection mechanism after homologous recombination of the donor DNA.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Endonucleasas/genética , Eubacterium/enzimología , Edición Génica/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Eubacterium/genética , Mutación PuntualRESUMEN
Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing.
Asunto(s)
Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Mutagénesis Insercional , Plásmidos/metabolismo , Transposasas/genética , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Electroporación , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Células HEK293 , Células HeLa , Humanos , Secuencias Invertidas Repetidas , Lípidos/química , Plásmidos/química , Análisis de Secuencia de ADN , Transfección , Transposasas/metabolismoRESUMEN
Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The 'reverse' reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase-RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase-RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , Recombinación Genética , Serina/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Sitios de Ligazón Microbiológica/genética , Bacteriófagos/genética , Fusión Génica , Integrasas/genética , Modelos Genéticos , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina/genética , Proteínas Virales/genéticaRESUMEN
Plants are a tremendous source of diverse chemicals, including many natural product-derived drugs. It has recently become apparent that the genes for the biosynthesis of numerous different types of plant natural products are organized as metabolic gene clusters, thereby unveiling a highly unusual form of plant genome architecture and offering novel avenues for discovery and exploitation of plant specialized metabolism. Here we show that these clustered pathways are characterized by distinct chromatin signatures of histone 3 lysine trimethylation (H3K27me3) and histone 2 variant H2A.Z, associated with cluster repression and activation, respectively, and represent discrete windows of co-regulation in the genome. We further demonstrate that knowledge of these chromatin signatures along with chromatin mutants can be used to mine genomes for cluster discovery. The roles of H3K27me3 and H2A.Z in repression and activation of single genes in plants are well known. However, our discovery of highly localized operon-like co-regulated regions of chromatin modification is unprecedented in plants. Our findings raise intriguing parallels with groups of physically linked multi-gene complexes in animals and with clustered pathways for specialized metabolism in filamentous fungi.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromatina/química , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Histonas/genética , Familia de Multigenes , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Avena/genética , Avena/metabolismo , Cromatina/metabolismo , Mapeo Cromosómico , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/metabolismo , Redes y Vías Metabólicas , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantones/genética , Plantones/metabolismo , Triterpenos/metabolismo , Zea mays/genética , Zea mays/metabolismoRESUMEN
The fields of molecular genetics, biotechnology and synthetic biology are demanding ever more sophisticated molecular tools for programmed precise modification of cell genomic DNA and other DNA sequences. This review presents the current state of knowledge and development of one important group of DNA-modifying enzymes, the site-specific recombinases (SSRs). SSRs are Nature's 'molecular machines' for cut-and-paste editing of DNA molecules by inserting, deleting or inverting precisely defined DNA segments. We survey the SSRs that have been put to use, and the types of applications for which they are suitable. We also discuss problems associated with uses of SSRs, how these problems can be minimized, and how recombinases are being re-engineered for improved performance and novel applications.
Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , ADN/metabolismo , Ingeniería Genética/métodos , Animales , ADN/química , ADN/genética , ADN Nucleotidiltransferasas/genética , Regulación Enzimológica de la Expresión GénicaRESUMEN
Sterols have important functions in membranes and signaling. Plant sterols are synthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to cycloartenol. Plants also convert 2,3-oxidosqualene to other sterol-like cyclization products, including the simple triterpene ß-amyrin. The function of ß-amyrin per se is unknown, but this molecule can serve as an intermediate in the synthesis of more complex triterpene glycosides associated with plant defense. ß-Amyrin is present at low levels in the roots of diploid oat (Avena strigosa). Oat roots also synthesize the ß-amyrin-derived triterpene glycoside avenacin A-1, which provides protection against soil-borne diseases. The genes for the early steps in avenacin A-1 synthesis [saponin-deficient 1 and 2 (Sad1 and Sad2)] have been recruited from the sterol pathway by gene duplication and neofunctionalization. Here we show that Sad1 and Sad2 are regulated by an ancient root developmental process that is conserved across diverse species. Sad1 promoter activity is dependent on an L1 box motif, implicating sterol/lipid-binding class IV homeodomain leucine zipper transcription factors as potential regulators. The metabolism of ß-amyrin is blocked in sad2 mutants, which therefore accumulate abnormally high levels of this triterpene. The accumulation of elevated levels of ß-amyrin in these mutants triggers a "superhairy" root phenotype. Importantly, this effect is manifested very early in the establishment of the root epidermis, causing a greater proportion of epidermal cells to be specified as root hair cells rather than nonhair cells. Together these findings suggest that simple triterpenes may have widespread and as yet largely unrecognized functions in plant growth and development.
Asunto(s)
Avena/metabolismo , Ácido Oleanólico/análogos & derivados , Epidermis de la Planta/metabolismo , Raíces de Plantas/metabolismo , Triterpenos/metabolismo , Avena/genética , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Glucuronidasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Transferasas Intramoleculares/genética , Transferasas Intramoleculares/metabolismo , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Ácido Oleanólico/metabolismo , Filogenia , Epidermis de la Planta/citología , Epidermis de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saponinas/metabolismo , Transcriptoma/genéticaRESUMEN
Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.
Asunto(s)
Avena/genética , Genes de Plantas , Familia de Multigenes , Saponinas/metabolismo , Triterpenos/metabolismo , Acilación , Aciltransferasas/clasificación , Aciltransferasas/genética , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Antifúngicos/metabolismo , Antifúngicos/farmacología , Ascomicetos/patogenicidad , Avena/enzimología , Avena/metabolismo , Regulación de la Expresión Génica de las Plantas , Metilación , Metiltransferasas/clasificación , Metiltransferasas/genética , Metiltransferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Filogenia , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Saponinas/genética , Relación Estructura-Actividad , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by C31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. C31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.
Asunto(s)
Integrasas/metabolismo , Ingeniería Metabólica/métodos , Redes y Vías Metabólicas/genética , Recombinación Genética , Bacteriófagos/enzimología , Vías Biosintéticas/genética , Clonación Molecular/métodos , Orden Génico , Ribosomas/metabolismo , Biología Sintética/métodosRESUMEN
Integrases, such as that of the Streptomyces temperate bacteriophage ÏC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ÏC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ÏC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ÏC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.
Asunto(s)
Bacteriófagos/enzimología , Integrasas/metabolismo , Recombinación Genética , Bacteriófagos/genética , ADN Viral/genética , Integrasas/genéticaRESUMEN
CRISPR activation provides an invaluable tool for experimental biologists to convert correlations into causation by directly observing phenotypic changes upon targeted changes in gene expression. With few exceptions, most diseases are caused by complex polygenic interactions, with multiple genes contributing to define the output of a gene network. As such researchers are increasingly interested in tools that can offer not only control but also the capacity to simultaneously upregulate multiple genes. The adaptation of CRISPR/Cas12a has provided a system especially suited to the tightly coordinated overexpression of multiple targeted genes. Here we describe an approach to test for active targeting crRNAs for dFnCas12a-VPR, before proceeding to generate and validate longer crRNA arrays for multiplexed targeting of genes of interest.
Asunto(s)
Redes Reguladoras de Genes , Personal de Salud , Animales , Humanos , Activación Transcripcional , Herencia Multifactorial , Mutagénesis Sitio-Dirigida , Mamíferos/genéticaRESUMEN
We present direct-LIVE-PAINT, an easy-to-implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct-LIVE-PAINT with an actin-binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct-LIVE-PAINT can be used to image actin structures below the diffraction-limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.
Asunto(s)
Citoesqueleto de Actina , Actinas , Animales , Actinas/metabolismo , Unión Proteica , MamíferosRESUMEN
Escin is a mixture of over 30 glycosylated triterpenoid (saponin) structures, extracted from the dried fruit of horse chestnuts. Escin is currently used as an anti-inflammatory, and has potential applications in the treatment of arthritis and cancer. Engineered yeast would enable production of specific bioactive components of escin at industrial scale, however many saponins have been shown to be toxic to yeast. Here we report that a Saccharomyces cerevisiae strain specifically lacking the sterol C-5 desaturase gene ERG3, exhibits striking enhanced tolerance to escin treatment. Transcriptome analyses, as well as pre-mixing of escin with sterols, support the hypothesis that escin interacts directly with ergosterol, but not as strongly with the altered sterols present in erg3Δ. A diverse range of saponins are of commercial interest, and this research highlights the value of screening lipidome mutants to identify appropriate hosts for engineering the industrial production of saponins.