Tuesday, July 21, 2015

Living Off Nothing But Coffee

Can you imagine living off nothing but coffee? Some of us probably feel like we do at times, if not for the taste then for the buzz the caffeine brings. Caffeine makes us feel more alert because it structurally resembles a molecule called adenosine.
Caffeine and adenosine are like brothers from another mother.
 Adenosine accumulates in our brain the longer we stay awake, binding to its receptors to induce that sleepy feeling we all get after a long day. It is the body’s way of signaling to the brain that it has had enough and needs to shut down for a while. If you disagree with your body, the ingestion of caffeine can help. Due to their structural similarity, caffeine competes with adenosine for binding to adenosine receptors; however, adenosine receptors do not execute the signal to shut down when bound to caffeine. In other words, the body is trying to throw a pass to sleep but caffeine blocks the receiver.

Some people can’t get to their happy place without a cup of joe in the morning. By the way, the term “cup of joe” is likely to have originated from “cup of jamoke” - “jamoke” being a combination of locales noted for their coffee goodness, “Java” and “Mocha”.
But that’s not all. Caffeine also ramps up adrenaline production, which increases your heart and breathing rates, and primes your brain and muscles for action. You feel a boost from coffee because the caffeine blocks the signal to sleep and fools your body into thinking it is under attack.
Like other drugs, people can build up a tolerance to caffeine, requiring more and more of the drug just to achieve the sensation of that original buzz. And the road to addiction is a short one, indeed. People love their coffee so much that the threat of a shortage can send them into a panic, which is perfectly captured in this scene from Airplane II.


How much coffee can people safely consume? According to the FDA, 400 mg (4 cups of brewed coffee) per day appears to be safe for most healthy adults. While it is estimated to take about 140 cups (8 oz size) of coffee to kill, you can get there a lot quicker with pure caffeine powder. A single tablespoon can be lethal, prompting the FDA to issue this warning to consumers.

But there is a creature on Earth that can tolerate much, much more. In fact, it eats coffee beans for breakfast. And lunch. And dinner. And everything in-between. Amazingly, the coffee berry borer eats nothing but coffee beans!

The coffee berry borer is a small but devastating beetle that lays waste to coffee crops. It subsists solely on coffee, capable of drinking any Starbucks junkie under the table.

Scientists have recently discovered how the coffee berry pest can tolerate toxic levels of caffeine. Do they possess a special gene that can detoxify caffeine? Do they have receptors that don’t bind caffeine? No…evidently, the answer does not lie in the genome of the beetle, but in its gut.
Like most other living creatures, the coffee berry borer houses a microbiome in its intestinal system. Several species of bacteria, such as Pseudomonas fulva, that reside in the gut of coffee berry borers are wizards at breaking down caffeine. The gut bacteria from coffee berry borers found around the world were put into culture medium containing caffeine as the primary nutrient so researchers could identify which species grew the best in this condition. P. fulva was the most common; subsequently, this bacterial species was found to carry a gene known to degrade caffeine.

To further test this hypothesis, researchers gave the beetles antibiotics to deplete their intestinal microbiome. Beetles without their gut bacteria lost the ability to break down caffeine. When fed some P. fulva before their coffee bean diet, the beetles excreted no caffeine, indicating that they were able to detoxify it once again.

Assuming no adverse effects, ingestion of P. fulva might help humans break down caffeine. A better alternative to decaf?
The scientists speculate that altering the beetle’s microbiome might provide a new approach in the battle against this pest. However, antibiotics are a precious commodity in treating human disease, so this could be a reckless idea as the introduction of antibiotics into the field has the potential of generating resistant bacteria.

From an evolutionary perspective, the study serves as an example of how organisms can adapt to a new niche without genetic modification. By acquiring specific types of bacterial symbionts, the coffee berry borer is uniquely able to live off nothing but coffee.

In the video below, you can learn more about this research and similar studies being performed at Lawrence Berkeley National Laboratory:

Contributed by:  Bill Sullivan
Follow Bill on Twitter.

See the news release at ScienceDaily.
Ceja-Navarro, J., Vega, F., Karaoz, U., Hao, Z., Jenkins, S., Lim, H., Kosina, P., Infante, F., Northen, T., & Brodie, E. (2015). Gut microbiota mediate caffeine detoxification in the primary insect pest of coffee Nature Communications, 6 DOI: 10.1038/ncomms8618

Tuesday, July 14, 2015

Complexities Of Allergic Disease

Last time we discussed the main players involved in the immune response to allergens, in the reaction called Type I hypersensitivity. We know that hay fever and other allergies are a result of atopy, the genetic predisposition to mount excessive IgE-mediated immune responses. Atopy is derived from a Greek word that means unusual or out-of-place. Although the immune overreaction is indeed out of place, the prevalence of allergic disease in society is not. Approximately 25% of the world’s population suffers from allergies, making it one of the most common chronic diseases. Unfortunately, this number is actually increasing, so researchers are trying to understand the factors that contribute to allergic disease.

Advances in genome sequencing and the completion of the Human Genome Project have allowed scientists to use genome-wide association studies (GWAS) in attempts to identify certain disease-causing genes. While many candidate genes have been described for hay fever, each search appears to reveal additional candidates. It has become clear that hay fever is a complex disease, driven by genetics and environmental exposures, both pre- and postnatal. Because of this complexity, atopy does not follow a Mendelian model of inheritance, like eye or hair color.
To perform GWAS, researchers collect blood or tissue samples from individuals with the disease of interest and from symptom-free control subjects. In many allergy studies, the controls are within the same family, which helps tease apart genetic differences that might actually contribute to disease. This is helpful because 300,000 to 1 million changes in the DNA are tested. These changes called single nucleotide polymorphisms (SNPs), a type of mutation that indicates a single change in the DNA base pair. If certain SNPs appear more frequently in the individuals with the disease, they are said to be associated with that disease. Additional DNA sequencing is performed to determine the exact change, and then ideally that SNP is studied in the lab to understand the consequence of the change on cellular function.
Sometimes mutations give super-human agility, strength, or intelligence. Other times they set us up to have wild and potentially unnecessary symptoms like Beast’s blue fur. Or perhaps equally annoying, the itchy watery eyes, nose, and throat from hay fever that come from a super-human response to harmless allergens.
A few notable candidate genes have been identified as associated with hay fever or with higher levels of circulating IgE antibodies, as we learned is a hallmark of atopy. Cytokines are the main signaling molecules that trigger activation of B cells to produce IgE antibodies, and not surprisingly, people with hay fever have mutations in genes that encode for cytokines or regulate their production. Also associated with hay fever are changes that enhance and stabilize the IgE receptor on mast cells and basophils, contributing to more intense symptoms. Genes responsible for airway smooth muscle contractions, contributing to cough and wheeze, are also implicated. SNPs have been identified in the genes encoding chemical mediators that cause ongoing symptoms such as leukotrienes, and in the specialized effector cells involved later in allergic inflammation response, such as eosinophils.

These are just a few examples of genes; dozens of others are being studied to learn exactly how they contribute to hay fever. Furthermore, some genes are only associated with allergies in the context of specific environments, further complicating the identification of true disease-causing genes. One clue that environmental factors influence allergies comes from studies of twins. Twin studies have shown that between monozygotic (identical) twins there is on average a 65% (range, 42-82%) chance that if one twin has allergies, the other will also have them. Between dizygotic (fraternal) twins, there is on average a 33% concordance rate (range 15-52%).
Some differences in twins are obvious, but other differences like allergies require epidemiological studies to tease apart.
The “hygiene hypothesis,” proposed by D. Strachan in 1989, became a popular basis to explore the increased incidence in allergic disease. Strachan observed that increased family size was associated with lower rate of allergies. He proposed that if allergies were prevented by early childhood infections, unhygienic contact with older siblings may protect against hay fever. Immune responses to pathogens like bacteria and viruses use a T-helper 1 (Th1) cell response. We know that Type I hypersensitivity reactions are mediated by Th2 cells’ stimulation of B cells to produce IgE antibodies. So the theory is that early childhood infections bias the immune system towards a Th1 response and suppress Th2 responses.

If Strachan’s hypothesis is correct, the Bates family should be allergen-free.
Strachan performed additional epidemiological studies to investigate the hypothesis that infections and larger family size protect against hay fever. He published a report in 2000 stating that decreases in family size do not appear to explain the increased incidence of allergies. Many additional studies looking into the protective effects of childhood infections have shown inconsistent results; some show a “protective” effect whereas others show either no association or early childhood infections correlated with development of allergies.
The “hygiene hypothesis” developed into a much broader “microflora hypothesis” which proposes that urbanization and a Western lifestyle limits our exposure to bacteria, viruses and parasites in general. Clean water, increased Cesarean sections, reduction in breastfeeding, increased antibiotic and antibacterial use, and reduced exposure to farm animals have limited our exposure to our microbial “old friends”. According to this idea, these “old friends” have evolved with us to the point where we require them for proper immune function.

“The Wonder Years” cast knew that we get by with a little help from our friends.

The diversity of our microbiome is decreasing, which may have detrimental effects on general health and the efficiency of our immune system. W. Parker proposed the term “biome depletion” to describe this current phenomenon. A few recommendations can be found here to increase microbial diversity in the gut.
Just like the Biodome, our biomes are not closed systems and can let in and respond to passer-byers, for better or for worse. In the case of Pauly Shore, it’s always for the worse.
If there weren’t already enough factors to consider in development of allergies, let’s peel back another layer. In addition to acquiring genes and microbiota from mothers during birthing and breastfeeding, in utero we are largely influenced by our mother’s environment through epigenetics. We’ve discussed epigenetics previously; briefly, it describes changes in the DNA and DNA-associated structural proteins that act to turn genes on or off. Epigenetic regulation either gives a green light or red light to production of specific gene products, or can act as a volume knob to finally tune gene expression. The process is plastic, allowing our genes to respond to the present environment.

Upon conception, epigenetic reprogramming occurs in the zygote, like a reset button. However, some epigenetic marks remain and are inherited by the offspring. So before and largely during pregnancy, the mother encounters various environments and the body responds using epigenetics to regulate genes at the appropriate time. These changes occur in the embryo or fetus as well, as a way to prime the baby for its eventual environment.
There is evidence that the immune system is under epigenetic regulation. At birth, atopy-prone infants tend to have diminished Th1 cell responses, thought to be influenced by the maternal environment. Additionally, maternal diet, microbial exposure, and smoking can influence epigenetic regulation of key genes involved in immune regulation and allergy development.
While there is no consensus on allergy prevention, there are many options for treatment of allergies, which will be discussed in the final allergy article in this series – coming soon!

Contributed by:  Julia van Rensburg, Ph.D.
Dávila I, Mullol J, Ferrer M, Bartra J, del Cuvillo A, Montoro J, Jáuregui I, Sastre J, & Valero A (2009). Genetic aspects of allergic rhinitis. Journal of investigational allergology & clinical immunology, 19 Suppl 1, 25-31 PMID: 19476051

Grammatikos AP (2008). The genetic and environmental basis of atopic diseases. Annals of medicine, 40 (7), 482-95 PMID: 18608118

Strachan DP (2000). Family size, infection and atopy: the first decade of the "hygiene hypothesis". Thorax, 55 Suppl 1 PMID: 10943631

Parker W (2014). The "hygiene hypothesis" for allergic disease is a misnomer. BMJ (Clinical research ed.), 348 PMID: 25161287

Martino D, & Prescott S (2011). Epigenetics and prenatal influences on asthma and allergic airways disease. Chest, 139 (3), 640-7 PMID: 21362650

Tuesday, July 7, 2015

The Anti-Vaccine Movement Is A Preventable Disease

Once again, vaccination is back in the news. On the heels of a new law passed in California that requires children to be vaccinated in order to attend schools and daycare centers, a woman in Washington has died of measles, marking the first death from this disease since 2003. The law also spurred shortsighted tweets from celebrities who think they are infectious disease experts, such as Jim Carrey (Dumb and Dumber, no pun intended), all of which have been thoroughly debunked. In light of this series of recent events, we're re-running Dr. Mark Lasbury's post on the anti-vaccine movement - consider it a "booster" shot. --Bill Sullivan


Scientific data can be interpreted many ways. That’s why scientists have conferences, to hash out the different reasons behind some observation…. plus the buffets and travel. One of the most famous arguments over interpretation of data was a series of debates between Albert Einstein and Nils Bohr at the Solvay conferences concerning the nature of quantum mechanics.

Bohr side held that at a quantum level, particles were undetermined until observed; the quantum world was one of probabilities, not observable realities. Einstein retorted, “God does not play with dice.” They both had the same data and they both agree on the data – it was the interpretation that was being debated (Bohr turned out to be right).

Because of multiple interpretations, science doesn’t like to say, “A caused B,” or even, “A correlates with B” unless they have the data to prove it. Premature interpretation can lead to bad data, bad hypotheses, and bad conclusions. And since so much of our culture today is based in science, bad conclusions lead to bad policy.

This means that when NO data exists to support a hypothesis, scientists have a duty to speak out against those making wild assertions. Especially when it leads to dangerous conclusions and actions. Vaccines causing autism is such a situation.

Just this week another scientific paper in the vaccine/autism controversy was withdrawn. A biochemical engineer named Hooker took data from an older CDC study and reinterpreted it. The CDC study had found no link between MMR vaccine and development of autism in children around two years old. They had looked at many factors to see if it occurred in subsets of patients as well, including race. But Hooker’s reanalysis showed that early MMR vaccination in African American boys was related to higher incidence of autism.

Lies, damn lies, and statistics. Do you really think organic
foods cause autism? Well there’s the data. CORRELATION
The paper from Hooker was withdrawn by the editors of the very obscure journal Translational Neurodegeneration because there are serious questions about his methodology and conclusions. Many scientists argue that if you parse the data in enough ways and do it enough times, you will always find a statistically significant number somewhere. As Gregg Easterbrook of the Atlantic Monthly said, “Torture numbers enough and they’ll confess to anything.” The question is whether it actually means anything.

For Hooker (the father of an autistic boy who he describes as "vaccine injured"), this meant taking a case control set of data and running a different style of study with the data (a cohort study). Also, his choice of statistical tests was wrong and he ended up comparing diagnosis at different ages instead of vaccination age versus diagnosis. Of course he found more autism diagnosis at 36 months than at 24 months – autism isn’t usually diagnosed until about 36 months! A great discussion of flaws in this paper is found here.

The vaccine/autism controversy started in the 1980’s, when the Urabe strain of mumps was being used in the MMR. But this was related to other brain lesions, not autism. Andrew Wakefield really got the ball rolling in a Lancet article in 1998. His conclusion was that the triple vaccine was the culprit and it should be given one at a time – anecdotally, the parent so his 12 subjects had said that autism started within days of their vaccination around 14 months.

Thimerosal used to be used as a preservative in vaccines.
It is 42% mercury by weight, and this used to be blamed
for autism. However, even though many studies have
shown it was not harmful, it is no longer used in
vaccines in the West.
Wakefield followed the idea that the MMR should be given separately with papers in 2001 and 2002 that said vaccination should be stopped and that vaccine had been found in the gastrointestinal tissues of children he said had “autistic enterocolitis.” But the papers included no new data, they were just rehashes of his initial paper with more dire conclusions. The media picked on these and more than 1500 articles were written about the “controversy” in 2002.

His co-authors withdrew their names when it became apparent that something was hinky – he was getting paid by a law firm that was suing the vaccine manufacturers (to the tune of a half million dollars). But he stuck by his guns. Then it was discovered that he was a major investor in a company ready to roll out vaccine-alternative products.

Then his data was reviewed in 2009 and was found fraudulent. He had manipulated data, including changing tissue sample results and making up parts of the histories of his child subjects. His license to practice medicine was taken from him in 2010 and I can’t for the life of me figure out why he isn’t in jail.

Anti-vaccine proponents say that vaccination during the months before two years of age alters the development of the brain and predisposes to autism spectrum effects. However, all the data on brain development suggests that the alterations that lead to autism take place in utero either as a result of genetics, trauma, or toxins – long before vaccination takes place.

The news isn’t all bad with autism. The new paper concerning
mTor has an upside. An antibiotic called rampamycin can
restore mTor activity and bring back synaptic pruning. The
authors ay it is reducing autism symptoms in mice, including
adult mice. You see the needed reduction in neural connections
in the above cartoon is between 2 and 4 years of age – right when
autism is diagnosed.
This includes a very recent paper that links autism to defects in a protein (mTor) that works to cut back synaptic connections in the developing brain. Too many connections leads to altered brain function and all too often, autism. But these changes are prior to vaccination or around time of autism recognition.

Research using 1st birthday party videos shows that many children show signs and symptoms of autism by 12 months, many months before most parents see the signs and a full year before autism can be reliably diagnosed. This may be a reason that people anecdotally link vaccines and autism – parents note it about the time a good number of vaccines are given. Some parents see it later using videos and use this regression of ability to damn vaccines - but the mTor paper addresses this well.

Unfortunately, some parents have used the erroneous data to draw a dangerous conclusion – they shouldn’t vaccinate their children. This has led to outbreaks of whooping cough, measles, mumps and other preventable diseases.

You might say that it doesn’t matter; if some parents choose not to vaccinate, then they're only hurting their own kids - but that isn’t so. For a small percentage of the population, especially the young and old, vaccination may not work properly or completely. This doesn’t matter if everyone vaccinates, because the presence of the disease in the population will be so low that their chances of being exposed remain low.

Jenny McCarthy has a son with autism. One can’t blame her
for her initial conclusion that vaccines might have been
involved, since fraudulent papers were out there. Now we
know better, but she hasn’t altered her “view” (although
she claims she has). Too often, I think we look for
something to blame.
But when more people that choose not to vaccinate their children, the presence of the disease goes up. Those who were vaccinated but did not receive full immunity, elderly patients who have lost some of their immunity due to an age-related decline in immune function, and even those people who are immune suppressed due to cancer or immune related diseases then run a much higher risk of being exposed.

Therefore, the “policy” of non-vaccination by some parents is flawed, in that there's no evidence to support their reason for withholding the vaccine, and because it injures other people in the community. Not a big deal? Mumps in adolescent and adult males can lead to sterility.

That isn’t scary enough? Then maybe it will be your child who isn’t as protected as they should be and becomes one of the 150 people a year who die each year of complications due to chickenpox, just because little Johnny’s mother “concluded” that vaccines were dangerous.
Contributed by Mark E. Lasbury, MS, MSEd, PhD

Hooker, B. (2014). Measles-mumps-rubella vaccination timing and autism among young african american boys: a reanalysis of CDC data Translational Neurodegeneration, 3 (1) DOI: 10.1186/2047-9158-3-16

Tang G, Gudsnuk K, Kuo SH, Cotrina ML, Rosoklija G, Sosunov A, Sonders MS, Kanter E, Castagna C, Yamamoto A, Yue Z, Arancio O, Peterson BS, Champagne F, Dwork AJ, Goldman J, & Sulzer D (2014). Loss of mTOR-Dependent Macroautophagy Causes Autistic-like Synaptic Pruning Deficits. Neuron PMID: 25155956

Thursday, July 2, 2015

Why Should You Care How Bacteria Fight Viruses?

Regular readers have been learning a great deal about the human immune system thanks to our ongoing series on allergies by Julia van Rensburg. But did you know that bacteria have an immune system of sorts, too? Yes, even germs get germs!* Bacteria are susceptible to a group of viruses called bacteriophages, or phages for short. Phages resemble early spacecraft and “land” on the surface of bacteria in order to inject their DNA/RNA, much like a syringe ejects its contents.

Houston, we have a problem! A phage has just injected its DNA into our cell!
Bacteria, which have been on Earth for some 3.5 billion years, have had plenty of time to evolve defense mechanisms against predatory phages. Just like human viruses, phages are a most unwelcomed guest. They barge into the cell unannounced, “borrow” cellular components without asking, and then use them to make baby viruses until the cell becomes so engorged with viral progeny that it explodes, releasing the huge viral family so that it can invade more bacteria and repeat the process all over again. Phages that burst the bacterium like this are called “lytic”, but there are other types that don’t blow the house up. These are referred to as “lysogenic” phages and can insert their genetic material into the bacterial genome, becoming a permanent resident of that bacterium. Even more sinister, the incorporated viral genome is copied like all the other bacterial genes when the bacterium divides, so it is inherited by the daughter cell!

Lytic phages will replicate until they blow the infected bacteria apart. In contrast, lysogenic phages can stick around forever, even getting passed on to future generations since the viral genome was inserted into the bacterial genome.

So that sucks – imagine if you had uninvited viral DNA shoved into your DNA – such viruses basically transform you into a GMO. Sorry to inform you, but up to 8% of your genome is already littered with lots of viral DNA. If you oppose GMOs, I hope you can still stand to be in your own skin!

Presently, we don’t know how to remove foreign DNA from our own. But bacteria have figured out a way to get rid of incoming phage DNA, which provides the basis for a type of bacterial immune system.
Some combinations work great together, like chocolate and peanut butter. But getting viral DNA stuck into your own DNA, a strategy used by many viruses including HIV, is not a welcome combination.

In 1987, scientists uncovered unusual repeat sequences in the genome of E. coli bacteria, which were later named “clustered regularly interspaced short palindromic repeats”, or CRISPR. In the early 2000s, scientists identified bacterial proteins interacting with CRISPR sequences (now called CRISPR-associated (Cas) proteins) and discovered that they provide resistance to phage infection. Through the efforts of many laboratories, it is now known that bacteria can use a phage invasion as a vaccination by incorporating some of the foreign DNA between CRISPR repeat sequences. This provides the bacteria with a “catalogue” – a memory system, if you will – of foreign DNA that it can pass along to future generations.

But CRISPR is not just a storage system. The bacteria can retrieve these sequences and hook them to Cas9, a nuclease enzyme that can cut DNA. When foreign DNA enters that bacteria, its CRISPR-Cas9 system can specifically target the invasive element and neutralize it.

Foreign DNA, such as that injected by a phage, can be neutralized by CRISPR/Cas9, which serves as a type of bacterial immune system. Bacteria can store foreign DNA sequences in its genome and express them as crRNAs that bind to Cas9. If the bacterium encounters foreign DNA that matches any of the sequences stored in its CRISPR array, the crRNA will deliver Cas9 to that invading sequence to chop it up.

Pretty clever for tiny bacteria, huh? But here is where things get really interesting, or worrisome, depending on your appetite for paranoia. Scientists have adapted CRISPR/Cas9 to work in all sorts of cell types, including human. Cas9 acts as DNA shears that can cut wherever we tell it to by directing it with a “guide RNA” (analogous to how a crRNA operates in bacteria). This provides us with an unprecedented means to easily “edit” the genome of virtually any living thing, including stem cells and embryos. Furthermore, Cas9 has been modified to do more than just cut DNA; versions exist now that can insert new DNA sequences or switch out bad (mutated) DNA with good DNA.

In the hit TV show, Orphan Black, a group of clones discover that their DNA has been “barcoded” to designate them as intellectual property by their maker. Theoretically, CRISPR technology could have been used to tag DNA in this fashion.
The power of genome editing can be used for good. Several diseases, such as cystic fibrosis and sickle-cell anemia, are caused by a single mutation in one gene. CRISPR/Cas9 is a plausible tool that may be able to repair this defect. However, tinkering with one gene can have unforeseen repercussions on other genes, so this exciting technology could have adverse effects. In March, 2015, a group of scientists proposed a ban on editing the human genome, arguing that a greater understanding of how CRISPR/Cas9 works is required before we even consider applying it clinically.

Gene editing using CRISPR/Cas9 can be used to modify the genome of virtually any creature. One recent application is the creation of wheat that is resistant to a fungus that causes mildew.

Here is a video that shows how CRISPR/Cas9 works and some of the applications it may have down the road:

Contributed by:  Bill Sullivan
Follow Bill on Twitter.

*It should be noted that not all bacteria are “germs”; in fact, many species of bacteria inhabit our bodies to constitute our “microbiome” and provide important services to us. Learn more about your microbiome here.
Sander JD, & Joung JK (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature biotechnology, 32 (4), 347-55 PMID: 24584096

Garneau, J., Dupuis, M., Villion, M., Romero, D., Barrangou, R., Boyaval, P., Fremaux, C., Horvath, P., Magadán, A., & Moineau, S. (2010). The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA Nature, 468 (7320), 67-71 DOI: 10.1038/nature09523

Horie, M., Honda, T., Suzuki, Y., Kobayashi, Y., Daito, T., Oshida, T., Ikuta, K., Jern, P., Gojobori, T., Coffin, J., & Tomonaga, K. (2010). Endogenous non-retroviral RNA virus elements in mammalian genomes Nature, 463 (7277), 84-87 DOI: 10.1038/nature08695

Horvath, P., & Barrangou, R. (2010). CRISPR/Cas, the Immune System of Bacteria and Archaea Science, 327 (5962), 167-170 DOI: 10.1126/science.1179555

Baltimore, D., Berg, P., Botchan, M., Carroll, D., Charo, R., Church, G., Corn, J., Daley, G., Doudna, J., Fenner, M., Greely, H., Jinek, M., Martin, G., Penhoet, E., Puck, J., Sternberg, S., Weissman, J., & Yamamoto, K. (2015). A prudent path forward for genomic engineering and germline gene modification Science, 348 (6230), 36-38 DOI: 10.1126/science.aab1028