by Moore R, Spinhirne A, Lai MJ, Preisser S, Li Y1, Kang T1 Bleris L
"Controllable gene delivery via vector-based systems remains a formidable challenge in mammalian synthetic biology and a desirable asset in gene therapy applications. Here, we introduce a methodology to control the copies and residence time of a gene product delivered in host human cells but also selectively disrupt fragments of the delivery vehicle. A crucial element of the proposed system is the CRISPR protein Cas9. Upon delivery, Cas9 guided by a custom RNA sequence cleaves the delivery vector at strategically placed targets thereby inactivating a co-expressed gene of interest. Importantly, using experiments in human embryonic kidney cells, we show that specific parameters of the system can be adjusted to fine-tune the delivery properties. We envision future applications in complex synthetic biology architectures, gene therapy and trace-free delivery."
by Mitchell R. O’Connell,Benjamin L. Oakes,Samuel H. Sternberg,Alexandra East-Seletsky, Matias Kaplan& Jennifer A. Doudna
"The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage1, 2, 3, 4, 5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA4, 5, 6, 7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags."
"Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a "body of knowledge" from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community."
"The development of RNA-based devices called toehold switches that regulate translation might usher in an era in which protein production can be linked to almost any RNA input and provide precise, low-cost diagnostics."
by Medema MH, Cimermancic P, Sali A, Takano E, Fischbach MA
"Bacterial secondary metabolites are widely used as antibiotics, anticancer drugs, insecticides and food additives. Attempts to engineer their biosynthetic gene clusters (BGCs) to produce unnatural metabolites with improved properties are often frustrated by the unpredictability and complexity of the enzymes that synthesize these molecules, suggesting that genetic changes within BGCs are limited by specific constraints. Here, by performing a systematic computational analysis of BGC evolution, we derive evidence for three findings that shed light on the ways in which, despite these constraints, nature successfully invents new molecules: 1) BGCs for complex molecules often evolve through the successive merger of smaller sub-clusters, which function as independent evolutionary entities. 2) An important subset of polyketide synthases and nonribosomal peptide synthetases evolve by concerted evolution, which generates sets of sequence-homogenized domains that may hold promise for engineering efforts since they exhibit a high degree of functional interoperability, 3) Individual BGC families evolve in distinct ways, suggesting that design strategies should take into account family-specific functional constraints. These findings suggest novel strategies for using synthetic biology to rationally engineer biosynthetic pathways."
"Considerable work has focused on the control of gene expression, motivated by both a fundamental interest in biological research as well as by applications ranging from gene therapy to metabolic engineering.
Synthetic biology provides the platform and tools to design artificial regulators to control mRNA translation. In this work, we report a genetically encoded system to regulate mRNA translation using the Pumilio and FBF (PUF) domains in mammalian cells. PUF domain serves as a designable scaffold to recognize specific RNA elements, and the specificity can be altered easily to target any 8-nt RNA. In this system, the gene expression could be varied by over 17-fold when using PUF-based activators and repressors. The specificity of the method was established by using wild-type and mutant PUF domains.
Optogenetics is a technology that allows control of cellular events using visible light as the signal/inducer. We designed an optogenetic system that employs the light-sensitive dimerizing partners from Arabidopsis thaliana , Cryptochrome 2 (CRY2) and Cryptochrome-interacting basic-helix-loop-helix 1 (CIB1), to reconstitute an RNA binding peptide and a translation initiation protein, thereby activating target mRNA translation downstream of the binding sites. Moreover, the combination of the two technologies allows us to construct to a light-inducible gene expression system using PUF domains, which can be used to regulate cellular RNA functions in a light-sensitive manner.
Additionally, we found that PUF domains could also be used to repress mRNA translation in E. coli. Such a system adds an important tool of RNA/protein interaction into the repertoire of tools for genetic circuit construction in E. coli."
"The ability to perturb living systems is essential to understand how cells sense, integrate, and exchange information, to comprehend how pathologic changes in these processes relate to disease, and to provide insights into therapeutic points of intervention. Several molecular technologies based on natural photoreceptor systems have been pioneered that allow distinct cellular signaling pathways to be modulated with light in a temporally and spatially precise manner. In this review, we describe and discuss the underlying design principles of natural photoreceptors that have emerged as fundamental for the rational design and implementation of synthetic light-controlled signaling systems. Furthermore, we examine the unique challenges that synthetic protein technologies face when applied to the study of neural dynamics at the cellular and network level."
"For more than half a century chemists have searched for a plausible prebiotic synthesis of RNA. The initial advances of the 1960s and 1970s were followed by decades of measured progress and a growing pessimism about overcoming remaining challenges. Fortunately, the past few years have provided a number of important advances, including new abiotic routes for the synthesis of nucleobases, nucleosides, and nucleotides. Recent discoveries also provide additional support for the hypothesis that RNA is the product of evolution, being preceded by ancestral genetic polymers, or pre-RNAs, that are synthesized more easily than RNA. In some cases, parallel searches for plausible prebiotic routes to RNA and pre-RNAs have provided more than one experimentally verified synthesis of RNA substructures and possible predecessors. Just as the synthesis of a contemporary biological molecule cannot be understood without knowledge of cellular metabolism, it is likely that an integrated approach that takes into account both plausible prebiotic reactions and plausible prebiotic environments will ultimately provide the most satisfactory and unifying chemical scenarios for the origin of nucleic acids. In this context, recent advances towards the abiotic synthesis of RNA and candidates for pre-RNAs are beginning to suggest that some molecules (e.g., urea) were multi-faceted contributors to the origin of nucleic acids, and the origin of life."
A bacterium has been used to wish people a Merry Xmas. Grown by Dr Munehiro Asally, an Assistant Professor at the University of Warwick, the letters used to spell MERRY XMAS are made of Bacillus subtilis, a non-pathogenic bacterium which is found in soil and also human gut.
Paola Antonelli, Director of R&D and Senior Curator, Department of Architecture and Design, MoMA, introduces the symposium Synthetic Aesthetics: New Frontiers in Contemporary Design, an investigation of the intersections between synthetic biology and design.
The symposium features guest speakers David Benjamin, Daisy Ginsberg, Dan Grushkin, and William Shih.
"Start-up firms say robotics and software that autonomously record every detail of an experiment can transform the efficiency and reliability of research. Max Hodak has spent much of his academic career fixing the ways that scientists collect data. As a biomedical engineering student at Duke University in Durham, North Carolina, it frustrated him that his laboratory recorded its experiments in paper notebooks, leaving researchers to scour through the pages to find relevant data. So in 2008, he indexed all the notebook data on a computer and wrote a program to allow users to query it. “People were saying, 'Why are you wasting your time? That's not going to lead to publication,'” he recalls. But a year-and-a-half later, he returned to the lab from a stint in Silicon Valley to find that many of those earlier sceptics were now using his system. To Hodak, it was a sign that he should pursue his quest for efficiency in the lab. “I was always more interested in finding ways to do analysis more efficiently than in doing the actual analysis,” he says......"
If the controversy over genetically modified organisms (GMOs) tells us something indisputable, it is this: GMO food products from corporations like Monsanto are suspected to endanger health. On the other hand, an individual’s right to genetically modify and even synthesize entire organisms as part of his dietary or medical regimen could someday be a human right.
"The pioneering works of Watson, Crick, Wilkins, and Franklin [1,2] on the structure of DNA have captivated our imaginations for over half a century and continue to shape our future endeavors. The genetic code, a mystery for many years, was soon thereafter decoded by organic chemists employing organic synthesis of polynucleotides . Ever since, the construction of DNA has been central to our ability to probe the molecular nature of life. Synthetic biologists now push the limits of what can be engineered using DNA – from scratch if needed: complex genetic circuits, large metabolic pathways, and even whole genomes."
"Dr. Jay Keasling has biologically done what no one has been able to do chemically: cheaply and quickly synthesize an effective malaria medication (1).
Many of the 400 million individuals infected with malaria each year suffer because of a lack of effective, affordable therapies, especially after the malaria-causing Plasmodium parasite became resistant to often-used chloroquine-based drugs.
Artemisinin-based drugs are now faster acting and more effective. However, so far, the chemical has been laboriously sourced from the plant Artemisia annua, a type of wormwood. It appeared to be too expensive for large-scale use; that is, until Keasling recruited the tools of synthetic biology to coax yeast cells into making …"