by Asaf Levy,Moran G. Goren,Ido Yosef,Oren Auster,Miriam Manor,Gil Amitai,Rotem Edgar, Udi Qimron& Rotem Sorek
"CRISPR–Cas (clustered, regularly interspaced short palindromic repeats coupled with CRISPR-associated proteins) is a bacterial immunity system that protects against invading phages or plasmids. In the process of CRISPR adaptation, short pieces of DNA (‘spacers’) are acquired from foreign elements and integrated into the CRISPR array. So far, it has remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of self is mediated by the RecBCD double-stranded DNA break repair complex. Our results suggest that, in Escherichia coli, acquisition of new spacers largely depends on RecBCD-mediated processing of double-stranded DNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers both from high copy plasmids and from phages."
by Bogumil J. Karas,Rachel E. Diner,Stephane C. Lefebvre,Jeff McQuaid,Alex P.R. Phillips,Chari M. Noddings,John K. Brunson,Ruben E. Valas,Thomas J. Deerinck,Jelena Jablanovic,Jeroen T.F. Gillard,Karen Beeri,Mark H. Ellisman,John I. Glass,Clyde A. Hutchison III,Hamilton O. Smith,J. Craig Venter,Andrew E. Allen,Christopher L. Dupont& Philip D. Weyman
"Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research."
BioCoder is a quarterly newsletter for DIYbio, synthetic bio, and anything related.
Socrates Logos's insight:
Bioinformatics for Aspiring Synthetic Biologists
Edgar Andrés Ochoa Cruz, Sayane Shome, Pablo Cárdenas, Maaruthy Yelleswarapu, Jitendra Kumar Gupta, Eugenio Maria Battaglia, Alioune Ngom, Pedro L. Fernandes, and Gerd Moe-Behrens
"For a synthetic biologist or biohacker to be able to hack, design, create, and engi- neer biological systems, the ability to work with biological data is essential. Basic bioinformatics skills will be required in order to read, interpret, write, and gener- ate files containing DNA, RNA, protein, and other biological information. In this article, we will show the path you need to follow to implement a biological func- tion using online data. As a case study, we are using Imperial College’s 2014 iGEM project, which focused on the optimization of bacterial cellulose production for use in water filtration."
"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. "
*Chinese scientists genetically modify human embryos*
by David Cyranoski& Sara Reardon
"Rumours of germline modification prove true — and look set to reignite an ethical debate.
In a world first, Chinese scientists have reported editing the genomes of human embryos. The results are published1 in the online journal Protein & Cell and confirm widespread rumours that such experiments had been conducted—rumours that sparked a high-profile debate last month2, 3 about the ethical implications of such work.
In the paper, researchers led by Junjiu Huang, a gene-function researcher at Sun Yat-sen University in Guangzhou, tried to head off such concerns by using 'non-viable' embryos, which cannot result in a live birth, that were obtained from local fertility clinics. The team attempted to modify the gene responsible for β-thalassaemia, a potentially fatal blood disorder, using a gene-editing technique known as CRISPR/Cas9. The researchers say that their results reveal serious obstacles to using the method in medical applications.
"I believe this is the first report of CRISPR/Cas9 applied to human pre-implantation embryos and as such the study is a landmark, as well as a cautionary tale," says George Daley, a stem-cell biologist at Harvard Medical School in Boston. "Their study should be a stern warning to any practitioner who thinks the technology is ready for testing to eradicate disease genes.
Some say that gene editing in embryos could have a bright future because it could eradicate devastating genetic diseases before a baby is born. Others say that such work crosses an ethical line: researchers warned in Nature2 in March that because the genetic changes to embryos, known as germline modification, are heritable, they could have an unpredictable effect on future generations. Researchers have also expressed concerns that any gene-editing research on human embryos could be a slippery slope towards unsafe or unethical uses of the technique.
The paper by Huang's team looks set to reignite the debate on human-embryo editing — and there are reports that other groups in China are also experimenting on human embryos.
The technique used by Huang’s team involves injecting embryos with the enzyme complex CRISPR/Cas9, which binds and splices DNA at specific locations. The complex can be programmed to target a problematic gene, which is then replaced or repaired by another molecule introduced at the same time. The system is well studied in human adult cell and in animal embryos. But there had been no published reports of its use in human embryos.
Huang and his colleagues set out to see if the procedure could replace a gene in a single-cell fertilized human embryo; in principle, all cells produced as the embryo developed would then have the repaired gene. The embryos they obtained from the fertility clinics had been created for use in in vitro fertilization but had an extra set of chromosomes, following fertilization by two sperm. This prevents the embryos from resulting in a live birth, though they do undergo the first stages of development.
Huang’s group studied the ability of the CRISPR/Cas9 system to edit the gene called HBB, which encodes the human β-globin protein. Mutations in the gene are responsible for β-thalassaemia.
The team injected 86 embryos and then waited 48 hours, enough time for the CRISPR/Cas9 system and the molecules that replace the missing DNA to act — and for the embryos to grow to about eight cells each. Of the 71 embryos that survived, 54 were genetically tested. This revealed that just 28 were successfully spliced, and that only a fraction of those contained the replacement genetic material. “If you want to do it in normal embryos, you need to be close to 100%,” Huang says. “That’s why we stopped. We still think it’s too immature.”
His team also found a surprising number of ‘off-target’ mutations assumed to be introduced by the CRISPR/Cas9 complex acting on other parts of the genome. This effect is one of the main safety concerns surrounding germline gene editing because these unintended mutations could be harmful. The rates of such mutations were much higher than those observed in gene-editing studies of mouse embryos or human adult cells. And Huang notes that his team likely only detected a subset of the unintended mutations because their study looked only at a portion of the genome, known as the exome. “If we did the whole genome sequence, we would get many more,” he says.
Huang says that the paper was rejected by Nature and Science, in part because of ethical objections; both journals declined to comment on the claim (Nature’s news team is editorially independent of its research editorial team.)
He adds that critics of the paper have noted that the low efficiencies and high number of off-target mutations could be specific to the abnormal embryos used in the study. Huang acknowledges the critique, but because there are no examples of gene editing in normal embryos he says that there is no way to know if the technique operates differently in them.
Still, he maintains that the embryos allow for a more meaningful model — and one closer to a normal human embryo — than an animal model or one using adult human cells. “We wanted to show our data to the world so people know what really happened with this model, rather than just talking about what would happen without data,” he says.
But Edward Lanphier, one of the scientists who sounded the warning in Nature last month, says: "It underlines what we said before: we need to pause this research and make sure we have a broad based discussion about which direction we’re going here." Lanphier is president of Sangamo Biosciences in Richmond, California, which applies gene-editing techniques to adult human cells.
"At Advances in Genome Biology and Technology, a conference for genomic scientists held earlier this year, one speaker told attendees that the use of genome sequencing to improve patient care is no longer a far-off goal—it’s happening today. While you won’t encounter genome sequencing on an average visit to the ER, there are certain clinical areas where this technology has indeed become routine: cancer, pediatric care, the diagnosis and treatment of ultra rare diseases, and a few others.
During that same conference, other scientists and doctors recounted impressive cases, from leukemia patients whose cancer was accurately monitored with sequencing to epilepsy patients whose symptoms were successfully treated with a normally unrelated medication chosen because of DNA data. An infant with life-threatening liver failure was restored to health after emergency genome sequencing pinpointed the problem....."
"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...."
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