Scientists create mouse that resists cocaine's lure

By biochemically 'hard-wiring' synapses, they prevent drug addiction
Date:February 13, 2017
Source:University of British Columbia
Summary:Scientists have genetically engineered a mouse that resists addiction to cocaine.

This is a diagram showing synapses in the reward circuit of mice when exposed to cocaine: on left, a normal mouse, and on right, a mouse with increased levels of cadherin.   Credit: University of British Columbia

This is a diagram showing synapses in the reward circuit of mice when exposed to cocaine: on left, a normal mouse, and on right, a mouse with increased levels of cadherin.

Credit: University of British Columbia

Scientists at the University of British Columbia have genetically engineered a mouse that does not become addicted to cocaine, adding to the evidence that habitual drug use is more a matter of genetics and biochemistry than just poor judgment.

The mice they created had higher levels of a protein called cadherin, which helps bind cells together. In the brain, cadherin helps strengthen synapses between neurons -- the gaps that electrical impulses must traverse to bring about any action or function controlled by the brain, whether it's breathing, walking, learning a new task or recalling a memory.

Learning -- including learning about the pleasure induced by a stimulant drug -- requires a strengthening of certain synapses. So Shernaz Bamji, a Professor in the Department of Cellular and Physiological Sciences, thought that extra cadherin in the reward circuit would make their mice more prone to cocaine addiction.

But she and her collaborators found the opposite to be true, as they explain in an article published today in Nature Neuroscience.

Dr. Bamji and her collaborators injected cocaine into mice over a number of days and immediately placed in a distinctly decorated compartment in a three-room cage, so that they would associate the drug with that compartment. After several days of receiving cocaine this way, the mice were put into the cage and allowed to spend time in any compartments they preferred. The normal mice almost always gravitated to the cocaine-associated compartment, while the mice with extra cadherin spent half as much time there -- indicating that these mice hadn't formed strong memories of the drug.

To understand that unexpected result, Dr. Bamji and her associates in UBC's Life Sciences Institute analyzed the brain tissue of the genetically engineered mice.

They found that extra cadherin prevents a type of neurochemical receptor from migrating from the cell's interior to the synaptic membrane. Without that receptor in place, it's difficult for a neuron to receive a signal from adjoining neurons. So the synapses don't strengthen and the pleasurable memory does not "stick."

"Through genetic engineering, we hard-wired in place the synapses in the reward circuits of these mice," says graduate student Andrea Globa, a co-lead author with former graduate student Fergil Mills. "By preventing the synapses from strengthening, we prevented the mutant mice from 'learning' the memory of cocaine, and thus prevented them from becoming addicted."

Their finding provides an explanation for previous studies showing that people with substance use problems tend to have more genetic mutations associated with cadherin and cell adhesion. As studies such as this one illuminate the biochemical underpinnings of addiction, it could lead to greater confidence in predicting who is more vulnerable to drug abuse -- and enable people to act on that knowledge.

Unfortunately, finding a way of augmenting cadherin as a way of resisting addiction in humans is fraught with pitfalls. In many cases, it's important to strengthen synapses -- even in the reward circuit of the brain.

"For normal learning, we need to be able to both weaken and strengthen synapses," Dr. Bamji says. "That plasticity allows for the pruning of some neural pathways and the formation of others, enabling the brain to adapt and to learn. Ideally, we would need to find a molecule that blocks formation of a memory of a drug-induced high, while not interfering with the ability to remember important things."

Story Source:

Materials provided by University of British ColumbiaNote: Content may be edited for style and length.

Journal Reference:

  1. Fergil Mills, Andrea K Globa, Shuai Liu, Catherine M Cowan, Mahsan Mobasser, Anthony G Phillips, Stephanie L Borgland, Shernaz X Bamji. Cadherins mediate cocaine-induced synaptic plasticity and behavioral conditioningNature Neuroscience, 2017; DOI: 10.1038/nn.4503

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CRISPR Applications in Plants

A Report from the Plant and Animal Genomics Conference
DeeAnn Visk, Ph.D.

Recent work by University of North Carolina researchers in the model organism Arabidopsis shows that the CRISPR/Cas9 system correctly targets the desired loci in plant genomes. [AnRo0002/Wikimedia]

Recent work by University of North Carolina researchers in the model organism Arabidopsis shows that the CRISPR/Cas9 system correctly targets the desired loci in plant genomes. [AnRo0002/Wikimedia]

Are you a food label reader?  If so, you may have noticed some of your favorite snacks bear the phrase “partially produced with genetic engineering.” This makes sense, given that the soy lectin and corn syrup used in many foods is probably isolated from plants genetically modified to be resistant to a powerful herbicide, glyphosate. Genes, originally isolated from bacteria, were inserted into crop plants, conferring glyphosate tolerance to the soybeans, corn, and other crops. Then, federal regulations followed: requiring that human food made with these plants be labeled “partially produced with genetic engineering.”

While these genetically modified plants have been around almost 20 years, new tools for plant biologists have yielded new traits for plants. At the Plant and Animal Genomics Conference held recently in San Diego, a topic of great interest was applications of the CRIPSR/Cas9 system to plants.

One brilliant approach to using CRISPR in plants is to edit the family of genes that confers susceptibility to bacterial blight in rice. Bacterial blight in rice, caused by Xanthomonas oryzae pv. oryzae, is a huge problem in Asia and Africa.

“To understand sensitivity to bacterial blight, it is necessary to first understand the biology of the disease process,” explains Bing Yang, Ph.D., associate professor in genetics, development and cell biology at Iowa State University.

“Bacteria that cause the blight have effector proteins (called TALs; transcription activator-like) that transcriptionally activate a family of genes in rice, referred to as SWEET genes. We strategized that by mutating the promoter region of the SWEET family of genes, the bacterial TAL proteins would no long be able to bind to the promoter. Being unable to bind to the promoter DNA, the bacterial TAL proteins cannot induce expression of the SWEET genes. Hence, TAL proteins could no longer bring about a state of disease susceptibility in rice,” explains Dr. Yang.

“CRISPR experiments can be designed to leave no fingerprint, or exogenous DNA in the plants. From a regulatory standpoint, the USDA should accept rice plants with small deletions or mutations in their genomes as safe for field tests,” concludes Dr. Yang.

Using a similar approach, disease-resistant citrus trees have also been developed. In Florida, the citrus industry faces disease challenges from citrus canker and citrus greening disease caused by two bacteria, Xanthomonas citri and Candidatus Liberibacter asiaticus, respectively.

"Citrus canker is also a big problem," asserts Nian Wang, Ph.D., associate professor, department of microbiology and cell science, Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida. "A specific effector protein from the infecting bacteria binds to the promoter region of the canker susceptibility gene CsLOB1 to induce disease symptoms. By utilizing CRISPR techniques, we can target the promoter region or the coding region of the citrus susceptibility gene to mutate it in such a way to prevent binding of bacterial transducers."

The CRISPR/Cas9 system can be applied in a manner that leave no exogenous DNA in the citrus, which is very beneficial in getting USDA approval.

"Applying the same strategy for citrus greening disease, we have begun research to identify the key virulence factors and their targets," continues Dr. Wang. "We are mutating the putative targets using the CRISPR technology. We hope to generate citrus trees resistant to citrus greening disease."

Another talk at the conference was on gene editing in cereals by Ming Luo, Ph.D., of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra, Australia. Wheat rust is a huge problem in failure of wheat crops worldwide; finding a solution to the problem would be a milestone in addressing world hunger.

“A pilot study of CRISPR efficacy in rice was successful with a knockout of two closely linked genes. In contrast, the homologous CRISPR experiment in wheat did not lead to any mutations,” declares Dr. Luo. “In contrast, using TALEN in wheat yielded results.

“While CRISPR works in rice and barley, CRISPR editing in wheat has not worked in our hands. We conclude that employing TALENs as a gene-editing tool in wheat is more efficient than CRISPR.”

One drawback to the CRISPR/Cas9 system in plants concerns off-target effects. To assess these effects in plants, whole genome sequencing is the current gold standard.

“Recent work in the model organism Arabidopsis, shows that the CRISPR/Cas9 system correctly targets the desired loci in plant genomes,” states Cara Soyars, University of North Carolina doctoral candidate. “This finding contrasts with off-target CRISPR effects in animals where off-target effects are a serious concern. Extrapolating this to other genera of plants, we postulate that modifications to the Cas9 protein to increase specificity of the binding site is not necessary in plants.”

“Plant genomes have many redundant genes. Hence, to effectively knockout a particular pathway of interest, many genes need to be knocked out,” continues Soyars. “Our lab, the Zachary Nimchuk lab, has developed a system that allows entire families of genes to be targeted in one experiment. While the system is predicted to increase the risk of off-target effects, we have shown with whole genome sequencing that there are very few or no off-target effects in Arabidopsis.

“One of our studies necessitated the targeting of 14 genomic loci at once. Using the multiplexed CRISPR/Cas9 system, we had a 33­–92% success rate. Whole genome sequencing also revealed that chromosomal translocation events are extremely rare after genome manipulation in Arabidopsis via CRISPR/Cas9.

“We really do not know why there is such a lower rate of off-target effects in plants when compared to animals,” clarifies Soyars. “Speculatively, plants use nonhomologous recombination; whereas animals employ homologous recombination when joining double DNA breaks. Perhaps differences in these repair mechanisms explain the difference in off target effects?”

One advantage of the CRISPR/Cas9 system is the applicability across a wide range of organisms. Editing carried out for research purposes does not require the same level of stringency as those for therapeutic applications. However, any plants or animals undergoing genome editing will need to be carefully vetted.

The regulatory body overseeing this is the Animal and Plant Health Inspection Service (APHIS), which is part of the USDA. APHIS released for comment a policy suggesting a path forward. For now, very small changes [like single base insertion or deletions (2–10 base pairs removed)] do not seem to be of much interest to APHIS.

“The ability to make these tiny changes at a very specific place in the genome is the result of using CRISPR/Cas9 technology in plants,” affirms Jeff Wolt, Ph.D., professor of agronomy at Iowa State University. “In the past, genetic additions to plants included either exogenous genes or even some of the machinery to get the modifications incorporated.

“Dr. Bing screened plants to select the edited gene of interest, while selecting against the inclusion of the CRISPR machinery. Dr. Bing confirmed this with lots of sequencing. His letter of inquiry to APHIS posed the question: will these rice plants be subject to regulation? APHIS responded that the material can be used without regulatory oversight.

“Plant researchers are moving forward cautiously, as the all the wonderful technology from previous methods of transgenic manipulation was not fully realized due to public push-back. We need to ensure that what we are doing is well-communicated and transparent,” expounds Dr. Wolt.

“Plant sciences have lagged behind in adopting new technologies for genome editing for a couple of reasons,” he continues. “First, funding levels are generally lower for plant researchers than studies involving animals. Second, the techniques used to change the genome must go through the cell walls of plants; in animals, especially cell lines, it is much simpler to get the components of CRISPR/Cas9 into the cells.”

 “Another reason many of the exciting applications of CRISPR in plants are not discussed as often as medical applications,” explains Mark Behlke, M.D, Ph.D., CSO of Integrated DNA Technologies, “is that the development of agricultural applications done by industry is confidential and is not published quickly, or at all. Also, working with crop plant genomes can be more complex than mammalian cells; as these species are often polyploid, which makes manipulation of their genomes more complicated.  Furthermore, plant genomes often have huge repetitive content.

“On the other hand,” Dr. Behlke continues, “advances in CRISPR/Cas9 technology has made genome manipulation accessible for just about any research lab in the world. One method that is especially promising is the use of a DNA-free system to perform genome engineering in plants. In this sort of system, the RNA guide is bound to recombinant Cas9 protein and added directly into cells as a ribonucleoprotein (RNP) complex, with no use of plasmids or other DNA-based expression cassettes.

“A delivery method of coating gold nanoparticles with plasmids and shooting them into whole animals has worked in cattle vaccinations (‘biolistics’). This approach is already being applied to plants, to get the Cas9 RNP complexes into cells through their tough cell walls,” concludes Dr. Behlke.

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The Synthetic Biology Era Is Here—How We Can Make the Most of It

We are entering an era of directed design in which we will expand the limited notion that biology is only the ‘study of life and living things’ and see biology as the ultimate distributed, manufacturing platform (as Stanford bioengineer, Drew Endy, often says). This new mode of manufacturing will offer us unrivaled personalization and functionality. 

New foods. New fuels. New materials. New drugs.

We’re already taking our first steps in this direction. Joule Unlimited has engineered bacteria to convert CO2 into fuels in a single-step, continuous process. Others are engineering yeast to produce artemisinin — a potent anti-malarial compound used by millions of people globally. Still other microbes are being reprogrammed to produce industrial ingredients, like those used in synthetic rubber.


If we look far enough, future bio-based industries will discard expensive, complicated industrial chemical syntheses that use high temperatures, high pressures and toxic catalysts in favor of cheaper, more resource-efficient and less toxic biochemical syntheses. 

We will do these things, and then we will exceed them. Or at least, that’s one (perhaps shamelessly optimistic) version of the future. Alternate perspectives, both pessimistic and realistic, ought to be considered too. There are many opportunities here — billion-dollar companies to be built, billion-person problems to be solved, critical ethical debates to be discussed in public, and policy prescriptions to be scrutinized.

So, how should we view the new world of synthetic biology? Let’s take a look. 

The Optimists 

For the optimists and dreamers amongst us, it’s tempting to believe that synthetic biology will surely usher in a fantastic world of abundance.

The optimists dream of longer, healthier lives enabled by intelligent systems that diagnose our diseases before symptoms appear. They long for truly personalized medicine. They anticipate CRISPR-enabled cures for genetic diseases, cancer and beyond. 

The optimists see synthetic biology as a burgeoning field with unmatched potential for human good — potential that’s only comparable to that of artificial intelligence. 

The Pessimists 

The pessimists and the cautious quietly quiver at this perspective. Because we humans identify very strongly with biology, some consider ‘engineering life’ to be unnatural, unethical and arrogant. 

The pessimists worry about how synthetic biology will affect our jobs, our sense of humanity and our ecosystems. They imagine a day when bio-terrorists can fabricate synthetic pathogens that can survive, multiply and cause deliberate harm to us.

The pessimists are concerned about unintended consequences . They feel that the potential for misuse and abuse is so great that the risks of synthetic biology (synbio) outweigh the benefits.

The Realists

The realists sit somewhere in the middle.

They see the world-changing potential of synthetic biology, yet they are aware of the hurdles that must be overcome before the fun stuff starts happening. They may ascribe to the optimist’s portrait of the future, but they remind us that first, we need to make biology easier to engineer and program . We need to develop standards for engineering life, abstractions for biological code and better ways of sharing experimental procedures so that reliable lab results can be replicated in labs around the world.

All three agree on one thing—we are moving fast into the synbio era. They’ve formed their opinion based on this simple fact. And they’re right. 

Forward Momentum Is Undeniable

 Predicting the trajectory of technology is riddled with complexity, but we are seeing movement on a number of different interrelated fronts in synthetic biology.

  • The development of the first programming language for living cells.
  • The arrival of CRISPR — a game-changing tool for cheap, easy genetic manipulation.
  • The colossal drop in the cost of reading DNA.
  • The emergence of IndieBio — the world’s first biotech accelerator that’s tempting postgrads away from academia into the startup world.
  • The explosion of iGem — an annual global student competition where students design, build and test biological devices that do useful things like biosensors that screen drinking water for pathogens or toxic metals.
  • The growth of the iGem Registry — a growing catalog of standard biological parts that engineers can lean on when designing biological circuits.
  • The formation of BioBricks — which works to make synbio an open and collaborative science that serves the public interest.
  • Startups like Amino Labs and Bento Labs that are developing easy-to-use, portable, laptop-size mini-labs equipped for real science — reading DNA and culturing friendly bacteria to make perfumes.
  • The broad maker movement that’s causing technical disciplines — from bioengineering to programming — to be more accessible, more inviting and less mysterious to everyone.

Beyond this, the increased media attention —both the sensationalist headlines and the useful pragmatic discussions are symptomatic of the upward trajectory of this field.

Collectively, all of these trends point to a future in which synthetic biology could be truly transformative in energy, healthcare, manufacturing, agriculture and beyond. But they also illustrate the fact that synthetic biology is still a young field.

This is where the realists have a point—we’re not there quite yet. And the more widely available and powerful the tech gets, the more we’ll want to sort out the rules of the road.

How hard we work today may well determine whether the future tends toward the immensely positive visions of the optimists or those scary pessimistic outcomes.

What Still Needs Doing to Make the Most of Synbio?

Here’s some of the work we still need to do.

  • We need to automate the process of designing and optimizing microbial strains.
  • We need to fully engage the public about the implications of these technologies.
  • We need to decide what our new relationship with biology will look like.
  • We need to explore which applications are acceptable and which are not.
  • We need to fully consider the effects certain synbio applications could have on issues like inequality and discrimination.

Like all powerful technologies, synthetic biology is inherently dual use — it can be used for human good or to threaten human safety. And because biology is involved, there is an added threat of multiplication and self-replication associated with certain biological weapons.

So yes, there is immense potential, and we should be excited about what this future might look like. And yes, we need the dreamers and the technical experts to push the boundaries of what is considered possible from a technical perspective (in a safe way). But in our quest for a better tomorrow, we must not overlook or evade the necessary ethical questions today. 


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