"Best known as a gene-editing system, CRISPR/Cas9 is also being used to edit the epigenome, turning on specific gene promoters and enhancers. The trick is to silence CRISPR/Cas9’s DNA-cutting mechanism. Instead, the CRISPR/Cas9 machinery is used to deliver an enzyme, an acetyltransferase, which adds artificial epigenetic marks to the DNA packaging proteins known as histones.
Gene-editing technologies have been used in several investigations of transcriptional regulation, but with mixed results. For example, some technologies intended for transcriptional control do not enzymatically modulate the chromatin state. They remodel the epigenome indirectly, and so they do not allow specific epigenetic markers to be evaluated...."
"Discovery expands potential CRISPR toolbox for treating genetic diseases in humans.
A tweak to a technique that edits DNA with pinpoint precision has boosted its ability to correct defective genes in people. Called CRISPR, the method is already used in the lab to insert and remove genome defects in animal embryos. But the genetic instructions for the machinery on which CRISPR relies — a gene-editing enzyme called Cas9 and RNA molecules that guide it to its target — are simply too large to be efficiently ferried into most of the human body’s cells.
This week, researchers report a possible way around that obstacle: a Cas9 enzyme that is encoded by a gene about three-quarters the size of the one currently used. The finding, published on 1 April in Nature, could open the door to new treatments for a host of genetic maladies (F. A. Ran et al. Nature http://dx.doi.org/10.1038/nature14299; 2015)...."
*Design criteria for synthetic riboswitches acting on transcription*
by Wachsmuth M, Domin G, Lorenz R, Serfling R, Findeiß S, Stadler PF, Mörl M.
"Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell."
"A method has been developed to produce and integrate single-stranded DNA into genomic locations in bacteria in response to exogenous signals. The system functions similarly to a cellular tape recorder by writing information into DNA and reading it at a later time. Much like other cellular memory platforms, its operation is based on DNA recombinase function. However, the scalability and recording capacity have been improved over previous designs. In addition, memory storage was reversible and could be recorded in response to analogue inputs, such as light exposure. This modular memory writing system is an important addition to the genomic editing toolbox available for synthetic biology."
Competing endogenous RNAs, which include mRNAs, transcribed pseudogenes, long noncoding RNAs (lncRNA), and circular RNA (circRNA), regulate other RNA transcripts by competing for shared microRNA
MicroRNA is a small non-coding RNA molecule containing about 22 nucleotides found in plants, animals, and some viruses, which functions in RNA silencing and post-transcriptional regulation of gene expression
Messenger RNA is a large family of RNA molecules that convey genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression
Pseudogenes are sections of a chromosome that are imperfect, dysfunctional copies of functional genes that have lost their protein-coding ability or are otherwise no longer expressed in the cell
Long noncoding RNA (lncRNA) comprises a large and diverse class of transcribed RNA molecules with a length of more than 200 nucleotides that do not encode proteins, and whose expression is developmentally regulated and that can be tissue- and cell-type specific
Circular RNA (circRNA) is a type of gene regulating noncoding RNA which, unlike the better-known linear RNA, forms a covalently closed continuous loop and that have not been shown to code for proteins
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length, that has many functions but is most notable in the RNA interference (RNAi) pathway where it interferes with the expression of specific genes with complementary nucleotide sequences..."
"A new type of foundry has moved into Boston Harbor, but it has no metal cutters or molten steel. In the 18,000-square-foot (1,672 square meters) facility, engineers churn out products ranging from scents and flavors to probiotics that fight antibiotic resistance. All of the custom-designed products come from an unlikely source: microorganisms.
Ginkgo Bioworks, part of the OS Fund, is one of a growing number of companies engineering technology with lessons from nature. Its founders are redesigning industrial engineering for a new generation — a manufacturing revolution powered by biology.
Synthetic biology goes mainstream
This nascent field, known as synthetic biology, is now at a place similar to where computers were in the 1950s and 1960s — slow, tedious and manual. But it is rapidly advancing and evolving with new technology: The industry is expected to reach $5.6 billion by 2018 — up from $1.9 billion in 2013.
Like many synthetic biology companies, Ginkgo's first commercially ready products are in the food and cosmetics industries, and they take a page from humanity's long history of culturing foods. Just like yeast is used to make wine and beer, scientists are using the natural processes of microorganisms to produce new flavors, nutrients and perfumes. ..."
"Synthetic biologists have a vision. Researchers in this young field, who build ‘devices’ from engineered genes and other molecular components, imagine a future in which products such as drugs, chemicals, fuels and food are manufactured by microbes. These devices could even be wired up to create cellular computers, much as electronic transistors are wired up to make microprocessors (see Nature http://doi.org/3fz; 2013).
But if the dream is to be realized, those components need to perform more consistently and be more reproducible than they are now, especially as they move from the lab bench to the biofactory. Unlike silicon-based electronic devices, synthetic organisms assembled from genetic components do not always have predictable properties — at least not yet.
On 31 March, representatives from industry, academic institutions and government met at Stanford University in California to launch the Synthetic Biology Standards Consortium, an initiative led by the US National Institute of Standards and Technology (NIST) to address issues preventing the field from reaching its potential.
“It’s the signal of a maturing industry,” says Patrick Boyle, who oversees the organism-design pipeline at Ginkgo BioWorks, a synthetic-biology company in Boston, Massachusetts. “As we get better at synthetic biology, we want to make sure we are comparing apples to apples.”
The standards push comes at a pivotal point for synthetic biology. Ginkgo BioWorks is one of several ‘foundries’ set up to mass produce organisms for various uses. ....."
"Time Lapse Movie by Jerome Bonnet et al. (Stanford) showing brightfield (left), control signal change over time (middle) and gate output (right) in individual cells operating a transcriptor-based genetic amplifier. "
DNA sequencing requires millions of short “barcode” chains to identify distinct samples. (Image: Flickr/Shaury Nash) DNA sequencing and synthesis are two sides of the same coin, the “read” and “write” functions of genetic material.
by Masoumeh Emadpour, Daniel Karcher and Ralph Bock
"Riboswitches are RNA sensors that regulate gene expression in response to binding of small molecules. Although they conceptually represent simple on/off switches and, therefore, hold great promise for biotechnology and future synthetic biology applications, the induction of gene expression by natural riboswitches after ligand addition or removal is often only moderate and, consequently, the achievable expression levels are not very high. Here, we have designed an RNA amplification-based system that strongly improves the efficiency of riboswitches. We have successfully implemented the method in a biological system for which currently no efficient endogenous tools for inducible (trans)gene expression are available: the chloroplasts of higher plants. We further show that an HIV antigen whose constitutive expression from the chloroplast genome is deleterious to the plant can be inducibly expressed under the control of the RNA amplification-enhanced riboswitch (RAmpER) without causing a mutant phenotype, demonstrating the potential of the method for the production of proteins and metabolites that are toxic to the host cell."
"Synthetic small RNA transcriptional activators can regulate gene transcription in Escherichia coli.
'Learn from nature and copy what it does' is one of the guiding principles in the laboratory of Julius Lucks at Cornell University, but in their recent work, the researchers developed a strategy that seemingly expands what nature has to offer.
“We want to leverage our ability to model and measure RNA structures to make gene networks,” says Lucks. His team focuses on transcriptional control, and they aim to have RNA inputs control RNA outputs without involving proteins such as transcription factors. “The big conceptual advantage of RNA over proteins is that you can do design,” explains Lucks. “We know a lot more about RNA folding than we do about protein folding.”
The strategy of the Lucks team has been to observe RNA design principles in nature, characterize their structure and then apply these designs to the engineering of genetic circuits. The limitation is that whereas nature very efficiently uses small RNAs to repress transcription, there are to date no known instances of small RNAs alone activating transcription. “But,” says Lucks, “if you want to build networks, you need to turn things on as well as off.”
Melissa Takahashi, a graduate student in the lab, first focused on characterizing the function of a natural RNA transcriptional repressor mechanism: a special sequence upstream of a gene's coding region that can form RNA structures that allow or prevent progression of the RNA polymerase. These structures are switchable: in one case transcription is stopped by a transcriptional terminator RNA hairpin, and in the other case transcription is allowed by an antiterminator sequence that sequesters the terminator and prevents the formation of the blocking hairpin. Takahashi looked at the structural transitions needed in order to undergo the switch from active to inactive transcription; she then came up with a strategy to invert this repression mechanism into one that activates transcription by adding yet another layer of structural transitions using a small transcription activating RNA (STAR)...."
Comment to: Creating small transcription activating RNAs
by James Chappell, Melissa K Takahashi & Julius B Lucks
"We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression." http://bit.ly/1HjhZW7
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