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A living programmable biocomputing device based on RNA
Can sense and analyze multiple complex signals in living cells for future synthetic diagnostics and therapeutics
July 28, 2017
“Ribocomputing devices” ( yellow) developed by a team at the Wyss Institute can now be used by synthetic biologists to sense and interpret multiple signals in cells and logically instruct their ribosomes (blue and green) to produce different proteins. (credit: Wyss Institute at Harvard University)
Synthetic biologists at Harvard’s Wyss Institute for Biologically Inspired Engineering and associates have developed a living programmable “ribocomputing” device based on networks of precisely designed, self-assembling synthetic RNAs (ribonucleic acid). The RNAs can sense multiple biosignals and make logical decisions to control protein production with high precision.
As reported in Nature, the synthetic biological circuits could be used to produce drugs, fine chemicals, and biofuels or detect disease-causing agents and release therapeutic molecules inside the body. The low-cost diagnostic technologies may even lead to nanomachines capable of hunting down cancer cells or switching off aberrant genes.
Biological logic gates
Similar to a digital circuit, these synthetic biological circuits can process information and make logic-guided decisions, using basic logic operations — AND, OR, and NOT. But instead of detecting voltages, the decisions are based on specific chemicals or proteins, such as toxins in the environment, metabolite levels, or inflammatory signals. The specific ribocomputing parts can be readily designed on a computer.
E. coli bacteria engineered to be ribocomputing devices output a green-glowing protein when they detect a specific set of programmed RNA molecules as input signals (credit: Harvard University)
The research was performed with E. coli bacteria, which regulate the expression of a fluorescent (glowing) reporter protein when the bacteria encounter a specific complex set of intra-cellular stimuli. But the researchers believe ribocomputing devices can work with other host organisms or in extracellular settings.
Previous synthetic biological circuits have only been able to sense a handful of signals, giving them an incomplete picture of conditions in the host cell. They are also built out of different types of molecules, such as DNAs, RNAs, and proteins, that must find, bind, and work together to sense and process signals. Identifying molecules that cooperate well with one another is difficult and makes development of new biological circuits a time-consuming and often unpredictable process.
Brain-like neural networks next
Ribocomputing devices could also be freeze-dried on paper, leading to paper-based biological circuits, including diagnostics that can sense and integrate several disease-relevant signals in a clinical sample, the researchers say.
The next stage of research will focus on the use of RNA “toehold” technology* to produce neural networks within living cells — circuits capable of analyzing a range of excitatory and inhibitory inputs, averaging them, and producing an output once a particular threshold of activity is reached. (Similar to how a neuron averages incoming signals from other neurons.)
Ultimately, researchers hope to induce cells to communicate with one another via programmable molecular signals, forming a truly interactive, brain-like network, according to lead author Alex Green, an assistant professor at Arizona State University’s Biodesign Institute.
Wyss Institute Core Faculty member Peng Yin, Ph.D., who led the study, is also Professor of Systems Biology at Harvard Medical School.
The study was funded by the Wyss Institute’s Molecular Robotics Initiative, a Defense Advanced Research Projects Agency (DARPA) Living Foundries grant, and grants from the National Institute of Health (NIH), the Office of Naval Research (ONR), the National Science Foundation (NSF) and the Defense Threat Reduction Agency (DTRA).
* The team’s approach evolved from its previous development of “toehold switches” in 2014 — programmable hairpin-like nano-structures made of RNA. In principle, RNA toehold wwitches can control the production of a specific protein: when a desired complementary “trigger” RNA, which can be part of the cell’s natural RNA repertoire, is present and binds to the toehold switch, the hairpin structure breaks open. Only then will the cell’s ribosomes get access to the RNA and produce the desired protein.
Wyss Institute | Mechanism of the Toehold Switch
Abstract of Complex cellular logic computation using ribocomputing devices
Synthetic biology aims to develop engineering-driven approaches to the programming of cellular functions that could yield transformative technologies. Synthetic gene circuits that combine DNA, protein, and RNA components have demonstrated a range of functions such as bistability, oscillation, feedback, and logic capabilities. However, it remains challenging to scale up these circuits owing to the limited number of designable, orthogonal, high-performance parts, the empirical and often tedious composition rules, and the requirements for substantial resources for encoding and operation. Here, we report a strategy for constructing RNA-only nanodevices to evaluate complex logic in living cells. Our ‘ribocomputing’ systems are composed of de-novo-designed parts and operate through predictable and designable base-pairing rules, allowing the effective in silico design of computing devices with prescribed configurations and functions in complex cellular environments. These devices operate at the post-transcriptional level and use an extended RNA transcript to co-localize all circuit sensing, computation, signal transduction, and output elements in the same self-assembled molecular complex, which reduces diffusion-mediated signal losses, lowers metabolic cost, and improves circuit reliability. We demonstrate that ribocomputing devices in Escherichia coli can evaluate two-input logic with a dynamic range up to 900-fold and scale them to four-input AND, six-input OR, and a complex 12-input expression (A1 AND A2 AND NOT A1*) OR (B1 AND B2 AND NOT B2*) OR (C1 AND C2) OR (D1 AND D2) OR (E1 AND E2). Successful operation of ribocomputing devices based on programmable RNA interactions suggests that systems employing the same design principles could be implemented in other host organisms or in extracellular settings.
First human embryo editing experiment in U.S. ‘corrects’ gene for heart condition
By Ariana Eunjung Cha August 2 at 1:00 PM
Scientists have successfully edited the DNA of human embryos to erase a heritable heart condition, cracking open the doors to a controversial new era in medicine.
This is the first time gene editing on human embryos has been conducted in the United States. Researchers said in interviews this week that they consider their work very basic. The embryos were allowed to grow for only a few days, and there was never any intention to implant them to create a pregnancy. But they also acknowledged that they will continue to move forward with the science, with the ultimate goal of being able to “correct” disease-causing genes in embryos that will develop into babies.
The experiment is the latest example of how the laboratory tool known as CRISPR (or Clustered Regularly Interspaced Short Palindromic Repeats), a type of “molecular scissors,” is pushing the boundaries of our ability to manipulate life, and it has been received with both excitement and horror.
The most recent work is particularly sensitive because it involves changes to the germ line — that is, genes that could be passed on to future generations. The United States forbids the use of federal funds for embryo research, and the Food and Drug Administration is prohibited from considering any clinical trials involving genetic modifications that can be inherited. A report from the National Academies of Sciences, Engineering and Medicine in February urged caution in applying CRISPR to human germ-line editing but laid out conditions by which research should continue. The new study abides by those recommendations.
This animation depicts the CRISPR-Cas9 method for genome editing – a powerful new technology with many applications in biomedical research, including the potential to treat human genetic disease or provide cosmetic enhancements. (Feng Zhang/McGovern Institute for Brain Research/MIT)
Shoukhrat Mitalipov, one of the lead authors of the paper and a researcher at Oregon Health & Science University, said that he is conscious of the need for a larger ethical and legal discussion about genetic modification of humans but that his team's work is justified because it involves “correcting” genes rather than changing them.
“Really we didn’t edit anything. Neither did we modify anything,” Mitalipov said. “. . . Our program is toward correcting mutant genes.”
Alta Charo, a bioethicist at the University of Wisconsin at Madison who is co-chair of the National Academies committee looking at gene editing, said that concerns about the work that have been circulating in recent days are overblown.
“What this represents is a fascinating, important and rather impressive incremental step toward learning how to edit embryos safely and precisely,” she said. However, “no matter what anybody says, this is not the dawn of the era of the designer baby.” She said that characteristics such as intelligence are influenced by multiple genes and that researchers don't understand all the components of how such characteristics are inherited, much less have the ability to redesign them.
The research involved eggs from 12 healthy female donors and sperm from a male volunteer who carries the MYBPC3 gene, which causes hypertrophic cardiomyopathy. HCM is a disease of the heart muscles that can cause no symptoms and remain undetected until it causes sudden cardiac death. There's no way to prevent or cure it, and it affects 1 in 500 people worldwide.
Around the time the sperm was injected into the eggs, researchers snipped out the gene that causes the disease. The result was far more successful than the researchers expected: As the embryo's cells began to divide and multiply, a huge number appeared to be repairing themselves by using the normal, non-mutated copy of the gene from the women's genetic material. In all, they saw that about 72 percent were corrected, a very high number. Researchers also noticed that there didn't seem to be any “off-target” changes in the DNA, which has been a major safety concern of gene-editing research.
Mitalipov said he hoped the technique could one day be applied to a wide variety of genetic diseases — more than 10,000 known disorders can be traced to a single gene — and that one of the team's next targets may be BRCA, which is associated with breast cancer.
The first published work involving human embryos, reported in 2015, was done in China and targeted a gene that leads to the blood disorder beta thalassemia. But those embryos were abnormal and nonviable, and there were far fewer than the number used in the U.S. study.
Juan Carlos Izpisua Belmonte, a researcher at the Salk Institute who is also a co-author on the new study, said that there are many advantages to treating an embryo rather than a child or an adult. When dealing with an embryo in its earliest stages, only a few cells are involved, while in a more mature human being there are trillions of cells in the body and potentially millions that must be corrected to eradicate traces of a disease.
Izpisua Belmonte said that even if the technology is perfected, it could deal with only a small subset of human diseases.
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“I don’t want to be negative with our own discoveries, but it is important to inform the public of what this means,” he said. “In my opinion the percentage of people that would benefit from this at the current way the world is is rather small.” For the process to make a difference, the child would have to be born through in vitro fertilization and the parents would have to know the child has the gene for a disease to get it changed. But the vast majority of children are conceived the natural way, and this correction technology would not work in utero.
For years, some policymakers, historians and scientists have been calling for a voluntary moratorium on the modification of the DNA of human reproductive cells. The most prominent expression of concern came in the form of a 2015 letter signed by Jennifer Doudna, the co-inventor of CRISPR-Cas9, Nobel laureate David Baltimore and 16 other prominent scientists. They warned that eliminating a genetic disease could have unintended consequences — on human genetics, society and even the environment — far into the future.
Mitalipov said he hopes regulators will provide more guidance on what should or should not be allowed.
Otherwise, he said, “this technology will be shifted to unregulated areas, which shouldn’t be happening.”