Abstract：
:Combinatorial metabolic engineering has been widely established for the development of efficient microbial cell factories to produce the products of interest by precisely regulating the expression levels of multiple genes simultaneously. Here, we report a novel multifunctional CRISPR system that enables simultaneous gene activation, repression, and editing (CRISPR-ARE) with a single Cas9-VPR protein for combinatorial metabolic engineering applications in Saccharomyces cerevisiae. Via gRNA engineering, we achieved orthogonal transcriptional regulations and genome editing using the nuclease active Cas9-VPR fusion protein, individually or in a combinatorial manner. After establishing a system for stable expression of multiple gRNAs on the same plasmid, we first demonstrated CRISPR-ARE for simultaneous mCherry activation, mVenus repression, and ADE2 disruption in a fluorescence reporter strain. Subsequently, we adopted CRISPR-ARE for simple and fast combinatorial metabolic engineering, which improved the production of α-santalene for 2.66-fold in a single step. Because of its simplicity and modularity, the developed CRISPR-ARE system could be applied for facile multifunctional metabolic engineering of microbial cell factories, particularly for which only a few CRISPR proteins have been characterized.
journal_name
ACS Synth Bioljournal_title
ACS synthetic biologyauthors
Dong C,Jiang L,Xu S,Huang L,Cai J,Lian J,Xu Zdoi
10.1021/acssynbio.0c00218subject
Has Abstractpub_date
2020-09-18 00:00:00pages
2252-2257issue
9issn
2161-5063journal_volume
9pub_type
杂志文章abstract：:Robust and precise ratio control of heterogeneous phenotypes within an isogenic population is an essential task, especially in the development and differentiation of a large number of cells such as bacteria, sensory receptors, and blood cells. However, the mechanisms of such ratio control are poorly understood. Here, ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00030
更新日期：2019-06-21 00:00:00
abstract：:Optogenetic control of endogenous signaling can be an important tool for probing cell behavior. Using the photoresponse of the LOV2 domain of Avena sativa phototropin 1, we developed analogues of kinase inhibitors whose activity is light dependent. Inhibitory peptides were appended to the Jα helix, where they potently...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/sb5001356
更新日期：2014-11-21 00:00:00
abstract：:While engineered chimeric antigen receptor (CAR) T cells have shown promise in detecting and eradicating cancer cells within patients, it remains difficult to identify a set of truly cancer-specific CAR-targeting cell surface antigens to prevent potentially fatal on-target off-tumor toxicity against other healthy tiss...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00175
更新日期：2019-10-18 00:00:00
abstract：:Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-exis...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00288
更新日期：2017-07-21 00:00:00
abstract：:As engineering foundations such as standards and abstraction begin to mature within synthetic biology, it is vital that genetic design automation (GDA) tools be developed to enable synthetic biologists to automatically select standardized DNA components from a library to meet the behavioral specification for a genetic...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400135t
更新日期：2014-08-15 00:00:00
abstract：:Although recent advances in synthetic biology allow us to produce biological designs more efficiently than ever, our ability to predict the end result of these designs is still nascent. Predictive models require large amounts of high-quality data to be parametrized and tested, which are not generally available. Here, ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00204
更新日期：2017-12-15 00:00:00
abstract：:A streamlined approach toward the rapid fabrication of streptavidin-biotin-based protein microarrays was investigated. First, using our engineered versatile plasmid (pBADcM-tBirA) and an optimal coexpression strategy for biotin ligase and biotin acceptor peptide (BAP) chimeric recombinant protein, an autogeneration sy...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00343
更新日期：2020-09-18 00:00:00
abstract：:Dramatic improvements to computational, robotic, and biological tools have enabled genetic engineers to conduct increasingly sophisticated experiments. Further development of biological tools offers a route to bypass complex or expensive mechanical operations, thereby reducing the time and cost of highly parallelized ...
journal_title：ACS synthetic biology
pub_type： 信件
doi：10.1021/sb300054p
更新日期：2012-12-21 00:00:00
abstract：:The ATLAS of Biochemistry is a repository of both known and novel predicted biochemical reactions between biological compounds listed in the Kyoto Encyclopedia of Genes and Genomes (KEGG). ATLAS was originally compiled based on KEGG 2015, though the number of KEGG reactions has increased by almost 20 percent since the...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00052
更新日期：2020-06-19 00:00:00
abstract：:Cell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence sp...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00465
更新日期：2018-08-17 00:00:00
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journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00294
更新日期：2016-06-17 00:00:00
abstract：:Design and implementation of synthetic biological circuits highly depends on well-characterized, robust promoters with predictable input-output responses. While great progress has been made with heterotrophic model organisms such as Escherichia coli, the available variety of tunable promoter parts for phototrophic cya...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00505
更新日期：2020-04-17 00:00:00
abstract：:We developed a hybrid synthetic circuit that co-opts the genetic regulation of the native bacterial quorum sensing autoinducer-2 and imposes an extra external controller for maintaining tightly controlled gene expression. This dual-input genetic controller was mathematically modeled and, by design, can be operated in ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00179
更新日期：2020-10-16 00:00:00
abstract：:Living organisms employ endogenous negative feedback loops to maintain homeostasis despite environmental fluctuations. A pressing open challenge in Synthetic Biology is to design and implement synthetic circuits to control host cells' behavior, in order to regulate and maintain desired conditions. To cope with the hig...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00220
更新日期：2017-03-17 00:00:00
abstract：:Modern bioprocessing depends on superior cellular traits, many stemming from unknown genes and gene interactions. Tolerance to toxic chemicals is such an industrially important complex trait, which frequently limits the economic feasibility of producing commodity chemicals and biofuels. Chemical tolerance encompasses ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb400156v
更新日期：2014-07-18 00:00:00
abstract：:Understanding the molecular mechanisms underlying antibiotic resistance requires concerted efforts in enzymology and medicinal chemistry. Here we describe a new synthetic biology approach to antibiotic development, where the presence of tetracycline antibiotics is linked to a life-death selection in Saccharomyces cere...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.8b00121
更新日期：2018-08-17 00:00:00
abstract：:Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineerin...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00219
更新日期：2016-06-17 00:00:00
abstract：:This paper describes SBOLExplorer, a system that is used to provide intuitive searching within the SynBioHub genetic design repository. SynBioHub stores genetic constructs encoded in the SBOL data format. These constructs can represent genetic parts, circuits, and sequences. These constructs are often numerous, exist ...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00089
更新日期：2019-10-18 00:00:00
abstract：:Directed evolution requires the creation of genetic diversity and subsequent screening or selection for improved variants. For DNA mutagenesis, conventional site-directed methods implicitly utilize the Boolean AND operator (creating all mutations simultaneously), producing a combinatorial explosion in the number of ge...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00063
更新日期：2019-06-21 00:00:00
abstract：:An RNA-dependent RNA polymerase ribozyme that was highly optimized through in vitro evolution for the ability to copy a broad range of template sequences exhibits promiscuity toward other nucleic acids and nucleic acid analogues, including DNA, threose nucleic acid (TNA), and arabinose nucleic acid (ANA). By operating...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00044
更新日期：2019-05-17 00:00:00
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journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.5b00002
更新日期：2015-05-15 00:00:00
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journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00266
更新日期：2018-01-19 00:00:00
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journal_title：ACS synthetic biology
pub_type： 杂志文章
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journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.7b00113
更新日期：2017-09-15 00:00:00
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journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.9b00192
更新日期：2019-07-19 00:00:00
abstract：:The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of in vivo circuit behavior in the context of applications where the host microbes are...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/sb3000639
更新日期：2012-10-19 00:00:00
abstract：:Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min that causes delays in real-time detection of protein expression. Here, we engineer a genetically encoded f...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.0c00407
更新日期：2020-11-20 00:00:00
abstract：:Genetic switches in which the activity of T7 RNA polymerase (RNAP) is directly regulated by external signals are obtained with an engineering strategy of splitting the protein into fragments and using regulatory domains to modulate their reconstitutions. Robust switchable systems with excellent dark-off/light-on prope...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00248
更新日期：2017-02-17 00:00:00
abstract：:Ethylene is an industrially important compound, but more sustainable production methods are desirable. Since cellulosomes increase the ability of cellulolytic enzymes by physically linking the relevant enzymes via dockerin-cohesin interactions, in this study, we genetically engineered a chimeric cellulosome-like compl...
journal_title：ACS synthetic biology
pub_type： 杂志文章
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更新日期：2014-07-18 00:00:00
abstract：:A common approach to design genetic circuits is to compose gene expression cassettes together. While appealing, this modular approach is challenged by the fact that expression of each gene depends on the availability of transcriptional/translational resources, which is in turn determined by the presence of other genes...
journal_title：ACS synthetic biology
pub_type： 杂志文章
doi：10.1021/acssynbio.6b00361
更新日期：2017-07-21 00:00:00
