Gene editing and GMOs

This month, I'm tackling gene editing. It's finally time I read papers on the topic because I got an email advertisement announcing a new gene editing kit and I realized that I don't know the mechanistic details of the system (yes... There's science email spam...).

I'll be summarizing a 2014 review from the journal Cell. The title of the review is "Development and Applications of CRISPR-Cas9 for Genome Engineering" (unfortunately, the review is behind a paywall).

As you can imagine, gene editing is somewhat of a holy grail. To be able to erase undesired mutations in DNA would be a dream for many clinicians/doctors. But there are many different applications that don't necessarily have to do with erasing what we don't want, rather, we could introduce variations that we want: creating an animal model for a disease, developing crops with desired traits, etc.

The paper starts by providing a brief overview of how genome engineering has been done in the past (focusing primarily on mammalian cells), outlining the pros and cons of each method. The difficulties inherent in current methods has led to the development of programmable gene editing technologies, the most promising of which is the CRISPR-Cas9 system which is the focus of the paper.

Spouse: I need to clarify something before I continue. The system isn't quite as simple as I'll describe below. There are additional DNA/RNA sequences and enzymes that are part of the complex, and I'll be omitting them because I could hear your voice in my head complaining about all the acronyms and jargon. So consider this to be the uber-abridged Cliffs Notes on gene editing.

Cas9 is an endonuclease, meaning that it's an enzyme that can cut both strands of DNA's double helix. Endonucleases can be random, meaning that they can cut anywhere along the length of the DNA. In fact, one of the fears of scientists working with DNA is nuclease contamination, which can render your samples to DNA dust. Other endonucleases, such as restriction enzymes, search for a specific DNA sequence and cut at that site. However, the cut site cannot be specified and can be found frequently in a genome (i.e. restriction enzymes can cut thousands of times).

Unlike restriction enzymes, the bacterial Cas9 cuts at a specific site but the DNA sequence where it cuts can be specified. Cas9 is associated with the CRISPR system, which guides Cas9 to its target using a small piece of RNA. In nature, this small piece of RNA generally encodes for a viral (phage) sequence. Cas9 searches for the viral sequence and then hacks it up, which is why the CRISPR system is part of the bacteria's antiviral defense mechanism. However, the small piece of RNA that guides Cas9 can be replaced with a sequence of the researcher's choice. Part of the system's benefit is the fact that you can provide more than one guide molecule, meaning that you could direct the system to cut more than one place, if desired. The system can be specific in the DNA sequence that it cuts, however, as this paper highlights, off-target edits can occur and are an area of ongoing study/research.

The paper provides a history of the many discoveries surrounding CRISPR-Cas9. There are different mechanisms by which the CRISPR system gets activated, and after many years of research, it was decided that the CRISPR-Cas9 was the most promising in terms of trying to find a programmable system for gene editing. By 2013, researchers successfully engineered the CRISPR system from two types of bacteria, including the one used to make yogurt, to edit genes in mammalian cells.

So far, I've described how to get the system to cut where you want it to cut. But then what? If you think of editing as deleting something that's incorrect and typing in something else, how do you get "what's right" or "what you want" into DNA?

Once the DNA is cut, the cell's natural repair mechanism kicks in and one of two things can happen (see my bee-u-ti-ful graphic below):

• The two loose ends of the DNA get glued back together again. The system is error prone but easy to use, so if your goal is to create a protein that doesn't function or to delete it altogether, this may be the way to go. This process is known as non-homologous end joining.
• The break is detected by enzymes that look around for the proper template to use to fill in the gap. If that template is provided artificially, then it will copy in that sequence. The template that researchers provide can contain the desired sequence, additional sequence, etc. This process is known as homology directed repair.

CRISPR-Cas9 can also be modified so that the "search" function of the system remains intact, but the cutting function is disabled. As such, researchers can create a complex where they guide their enzyme of choice to a specific region. I think that the simplest example that the paper provides is one where Cas9 was fluorescently labelled/tagged, so researchers could visualize the location of the DNA sequence they were studying.

In reviewing this article, the spouse asked if we could write a movie script where the villian sprinkled Cas9 along with the DNA specific to his arch-nemesis' genome into the latter's cereal. Would it be the perfect crime? Would the CRISPR-Cas9 enter the arch-nemesis' body and hack up his DNA? Unfortunately, no. Keep in mind that our DNA is within the nucleus of our cells and isn't very accessible. Additionally, as non-bacterial species, we don't have CRISPR-Cas9 in our cells. Getting the CRISPR-Cas9 into the nucleus isn't all that simple and requires a bit of fancy lab work.

To date, there's no medical therapy on the market developed using gene editing. Likewise, there's no crop on the market that has been engineered using CRISPR-Cas9. However, studies have demonstrated that crops can be modified using the system (this paper provides an example of successful gene editing using CRISPR-Cas9 in rice). Consequently, many wonder whether crops generated through gene editing would be considered GMOs.

As I've previously described, what are currently known as a GMOs or genetically modified organisms are transgenic crops, meaning that a gene from a different species has been added to their genome (NOTE: in this post, I note that anti-GMO activists have a very different definition of a GMO). But in the case of crops modified using CRISPR-Cas9, what's edited was there to begin with. Technically, nothing has been added from a different species. So how will regulatory agencies categorize these crops?

This paper published just last month provides a great summary: it states that the USDA has concluded that if you cannot distinguish an edit from a naturally occurring mutation, then it's not a GMO. Additionally, if a gene is deleted using the cell's own repair mechanism (as is the case with non-homologous end joining), then it isn't a GMO either. Interestingly, the paper states that the USDA has waived regulations on two crops generated using gene editing, because they fell within these categories. The European Union has yet to determine how these crops will be classified, because they consider something to be genetically modified if "it is altered in a way that does not occur naturally by mating and/or natural recombination" (although crops generated through mutagenesis are not regulated in the EU. Please see my previous post on mutagenesis for more information on this technique). There are two additional points that the paper makes that I completely agree with: 1) if the EU's definition of a GMO does not end up aligning with the USDA's, the regulation of these crops for import will be very difficult since there will not be an easy way to detect if the crop is a product of gene editing. 2) If the EU's definition of a GMO does not end up aligning with the USDA's then the cost of getting a crop through regulatory hurdles will limit the development of these plants to large biotech companies, which will stifle innovation; i.e: if you want someone other than Monsanto, Syngenta, et al to make a biotech crop, these crops should not be considered GMOs.

To conclude, here's my first "infographic" on the different methods or ways used to develop new traits in crops and feedback would be appreciated. My perspective on gene editing is the same as it is on transgenesis and mutagenesis: crops should be regulated based on the trait introduced/modified, not on the way that the introduction/modification was generated.

Review of "GMO Myths and Truths" - Part 1

During a recent twitter exchange, I was sent a document entitled "GMO Myths and Truths: An evidence-based examination of the claims made for the safety and efficacy of genetically modified crops". The document was sent to me as evidence that many scientists are opposed to GMOs. I will be reviewing this 123 page document in its current version (1.3b), in several parts.

My delicious Urban Rabbit lunch salad

The document has three authors. The first author has a PhD in Molecular Genetics, is well published, and works on developing human gene therapies. The second author is an editor at GM Watch and a research director at Earth Open Source (who put together the document). The third author, has a PhD in molecular genetics, and is also the founder and CSO at a GMO testing and certification company. He is also the co-founder of Earth Open Source. Earth Open Source is an anti-GMO organization, whose website's main page includes the infamous "lumpy rat" pictures from Seralini's now-retracted article (for my views on the retraction of the study, please see here). I highlight the background of the authors to note that there are only 2 scientists, one of whom benefits financially from anti-GMO sentiments.

The first section of the document outlines that although proponents of GMOs state that the genetic engineering process is just an extension of natural breeding, is safer than mutagenesis, and more precise than traditional cross-hybridization, it actually isn't. They admit that the genetic engineering process has become better and more precise, but you can still cause unintended consequences. They list a whole slew of unintended consequences that could happen. The word "could" is key in this section.

Well, we've gotten better at a lot of different things over the past few decades. As mentioned, the authors admit that we've gotten better at creating transgenic crop, but they omit that we've gotten astronomically better at detecting unintended consequences. Technologies such as whole genome sequencing and microarrays are used because of the whole "we don't know what we don't know" phenomenon. These technologies will allow you to analyze RNA and DNA so that you can identify mutations that you didn't intend to make or RNA hybrids that you were unaware of. In a recent Q&A with the Arctic Apple company on GMO Skepti-Forum, Arctic Apple's staff mentioned that they had the genome of their apple sequenced (for a review of the Arctic Apple, please see here). All 750 million bases of it. And their conclusion was that there were no unintended mutations. So it's not surprising that in an online search, I was able to find out that companies such as Monsanto and Dow Agro use these technologies as well to study GMOs.

As I understand it, you make many different transgenic lines in the process of making a GMO and you test them to find out which ones are expressing the gene you're interested in at the level you desire and in an appropriate location. Yes, the authors of the document provide information on the many ways things could go wrong, but that's why a transgenic plant isn't made in a single shot. I sort of feel that this section in the document is like making a giant list of all the parts in your car that could break down and then concluding that you shouldn't drive your car... (Spouse, I'm reminded that my car is making a weird humming sound. Could you check it out?)

There are a few segments on mutagenesis and cis-genesis in the document, and I've previously reviewed this topic here (briefly, cis-genesis is taking a gene from the same species and modifying it. Mutagenesis is using chemicals/radioactivity to modify a trait within a species. Plants derived through mutagenesis are not considered GMOs, but are lumped under "conventionally bred organisms"). The authors are of the opinion that these technologies are just as risky as transgenesis, and should be regulated and tested. My personal opinion on this topic is that the technology used to generate the trait shouldn't matter, and that the degree of regulation/testing should depend on the trait itself.

Section 1 of the document boils down to a difference of opinion between these two authors and many other scientists. The authors conclude the first section stating that using genetic engineering to create new strains is unnecessary, because conventional breeding can meet crop breeding needs. Well, I'm not sure that's accurate, unless you discard transgenesis altogether, and I think that many scientists would disagree with this point of view. I feel that this whole section was like a side-by-side comparison of using a knife vs using a food processor, and then concluding that you don't need a food processor because the knife can do the same thing, but stressing all the additional risks when you use the food processor such as the possibility of getting electrocuted, shocked, etc. The document skipped over the benefits of the food processor and its strengths, and failed to highlight the drawbacks of a knife.

Section 2 questions the idea of "substantial equivalence". I have to read all the papers that they're citing, so I'll leave that for another day. For now, Baby Boy is being weaned off his pacifier and he's not happy about it, so I've gotta boot.

Getting E. coli poisoning from GMOs

I can't sleep. It's 1:00AM, and instead of tossing and turning and keeping my husband awake, I've opted for exhausting myself by writing.

This week's post is brought to you courtesy of Twitter. It all started when my twitter feed linked me to a doozy of a story, about how you can get E. coli poisoning from GMOs. The author outlines that E.coli is used during the course of genetic engineering to replicate DNA since it is highly prolific. But, the author highlights, it is also able to transfer DNA laterally to and from other species. Then the author says: "It is possible that a mutated form of e-coli resulting from the cloning process used in creating GMOs could get into the gut of a person or animal that eats a transgenic plant." The author then highlights that Dupont has partnered with the USDA in "identifying hard-to-identify strains of E.coli". The theory comes full circle with the statement: "with this convenient partnership, even if harmful strains of e-coli relating to GMOs are discovered, it is likely that the public will never hear it from the USDA or DuPont."

I feel like the author of this post missed out on an opportunity to write for the X-files a few years back.

I then spent two days with the individuals posting these articles trying to discover how this could happen, but it never when beyond "the YUK factor" of using E.coli in generating our food. I even offered a 1 hour tutorial to share information on E.coli's use in genetic engineering, which got turned down (I wonder how my previous customers would feel if they knew that I was offering consulting services FOR FREE on the interwebz). What I did notice was that there were a couple of comments that probably made great memes for someone which got retweeted a whole bunch of times. They were mostly about how our babies are being exposed to E.coli and how our GMO corn is contaminated with E.coli. YUK.

To quote one of my colleagues, "my biggest concern is the unpredictability of evolutionary mutability, and the off-target effect of E. coli-mediated GMO excrutabilation likely resulting in contamination/Monsantinization of our 1.21 gigawatt-phase diarrhea chute." Doesn't make sense? Well, neither does the spontaneous mutation of E.coli from GMOs.

Since my tutorial was turned down, I will write down a few facts about E.coli's use in genetic engineering here.

• E.coli is used in the lab specifically because the strain used IS NOT HARMFUL. The non-virulent nature of the bacteria isn't due to a mutation that might spontaneously arise to make it virulent again. There are entire genes that are different between virulent and non-virulent strains of E.coli. In fact, this paper that looked at 61 different strains of E.coli found that only 80% of their genomes are in common. That's much less than the >95% that we share in common with chimps.  
• E.coli is used in the lab because it grows like crazy. When it replicates it also copies its DNA.
• If you add the DNA that you're interested in studying to E.coli's DNA, then that will also replicate.
• Why would you do this? Well, many procedures in the lab require a lot of DNA (by "a lot", I actually mean microgram or nanogram quantities. But for a molecular biologist, that's a lot). So how else can you get that much DNA that you're interested in? The issue of amplifying and copying DNA is not unique to the process of making GMOs. So the technique of adding/removing DNA from E.coli, also known as cloning, is very common.
• Here's an extremely simplistic overview of E.coli cloning: to add/remove DNA, you add an enzyme that cuts your gene of interest (enzyme is known as "restriction enzyme"). Then you purify the piece that was cut. The way that your gene was cut will be in a specific pattern, similar to that of a puzzle piece. Then, you cut the bacterial DNA with the same enzyme so that the two puzzle pieces will fit together. The pieces get "glued" together with yet another enzyme known as a "ligase". The glued piece of DNA goes into the bacteria, which then replicates. A few hours later... voila!! You have lots of bacteria that have lots of your DNA of interest.
• But then, and here's the part that the authors of the lovely article above fail to mention, you have to get your DNA out of the E.coli. To get the DNA out of the bacteria, by definition, involves killing the bacteria. You pop the bacteria open, you clean up the goop, and you have bacterial DNA. THEN, you have to cut the DNA again so that you can get that piece of DNA that you were trying to amplify all along. So you leave the E.coli DNA behind, which again, was inconveniently left out in the article above. I've even made a pretty picture :)


• If you add DNA for entire genes to bacteria, sometimes they can actually produce that protein. This is the life-saving technology used to generate insulin, many synthetic vitamins, and drugs. In the case of insulin, the strain of bacteria used is E.coli. And despite my searches on the web, I've been unable to find a case of someone getting E.coli poisoning from insulin. Try telling a diabetic that there's a YUK factor when it comes to their insulin.

So, today (which, at 4AM is actually yesterday) is International Women's Day and I'm going to end with a very personal comment here: To my fellow women. To all the brave moms out there doing their best, just like me. You do the groundbreaking women in science an immense disservice by willingly choosing to remain ignorant on a topic and then perpetuating erroneous information. We women in science do what we do only because there was a Rosalind Franklin and a Nettie Stevens who paved the way for us. And you're dropping shards of glass on that path. I kid you not. When you're up-in-arms about babies being exposed to E.coli through GMOs, you spread misinformation, perhaps even prey on the susceptibilities of other moms, and you perpetuate decade-old stereotypes about gender. The specific one I refer to is depicted in this oft-used clip from the Simpsons, where Mrs Lovejoy appears in town-hall meetings where progressive topics are addressed screaming "Think of the children!" and pulling her hair out. The specific stereotype I refer to is that we are emotionally driven creatures who sacrifice logic and common-sense when it comes to the safety of our children. Aren't we supposed to be helping each other out? Aren't we supposed to be part of a brighter generation of women who can be moms AND be smart? So why is it that you willingly turn down knowledge and choose to spew gibberish about how "GMOs are changing our evolution"? To paraphrase a recent interview I saw with Neil deGrasse Tyson, I can't blame you if you are unaware of scientific facts and truths. That is probably just a factor of the education system that exists today. However (and this part is my own opinion, not Dr. Tyson's), I can blame you if someone tries to correct you and educate you on these scientific truths and you choose to ignore them.

So let me make this abundantly clear: there is NOTHING, let me repeat that: NOTHING, written in that article about the risks and dangers of E.coli's use in genetic engineering that is accurate. If you want to argue against GMOs, please use arguments that are evidence based and do not propagate scientific illiteracy. Think of the children!

IRT - GM food supplement caused deadly epidemic

Before I get started, I have a confession to make: I love pomegranates. Love probably isn't a strong enough word. I ate 2-3 a day while I was pregnant, to the point that the hubby planted two trees for me (one of which had it's first fruit this year). But it's a "work food". Like crab legs. Or pumpkin seeds. You have to put in a good amount of effort before you can eat the stuff. Anyway, since it's pomegranate season, I've been eating them all the time and I woke up thinking "someone should make a pomegranate that's easier to clean". And I'm guessing I'm not the only one who wishes for this: Wikipedia has a good section dedicated to suggestions on cleaning out pomegranates (including my preferred method of cracking them open under water). Well, according to this review, a genetically modified pomegranate tree is possible because one has already been made to express GFP (green fluorescent protein). It's probably what Sheldon used to make his goldfish glow in the dark. The authors recommend making pomegranate crops that are stress tolerant or have other horticulturally-beneficial genes, but none have been made (although that hasn't stopped people from marketing GMO-free pomegranate juice). None of the suggestions in the review included recommendations on making the fruit easier to eat...

Now, getting onto juicier topics (no pun intended!)

If you've been following this blog, you'll know that I started reviewing the "Health Risks" section on the Institute for Responsible Technology's webpage several months ago. This was due to the fact that numerous anti-GMO webpages were using it as a resource, so I thought I should look into it. Today, I finish its review by covering a section entitled "GM food supplement caused deadly epidemic". I'll be honest here: I started out upbeat and open minded when I started reviewing the IRT's site, but I haven't come across a single valid statement. Consequently, every time I sit down to read from this webpage, I feel the same way as when I think about Jar Jar Binks. Or just Episode 1 in general. A sense of dread and gloom. Consequently, it's only this bowl of pomegranate that I'm currently eating that will get me through this last section.

The segment would have been more aptly entitled "Dietary supplement produced using recombinant DNA technology was associated with a deadly epidemic". Here's the first section directly from the IRT: "In the 1980s, a contaminated brand of a food supplement called L-tryptophan killed about 100 Americans and caused sickness and disability in another 5,000-10,000 people. The source of contaminants was almost certainly the genetic engineering process used in its production." It goes on to state that it took years to "find" the disease and that an investigation only took place because the symptoms were so dire. The IRT argues that there is no monitoring of GMO-related illnesses - particularly long-term effects - and it may take decades, if ever, to identify the source of a problem. I've already reviewed the topic of long term effects of GMOs, so I'll focus on the first part of their argument.

Unfortunately, there are only two citations. The first is to an article on the IRT's webpage and the second is to a book written by one of the founders of the IRT. So neither one are peer reviewed sources. However, Wikipedia has a pretty good entry about this event, with several citations. The disorder is known as eosinophilia-myalgia syndrome (or EMS) and the outbreak took place in 1989, leading to an eventual ban of tryptophan dietary supplements by the FDA. I started by reading a few abstracts for papers that investigated the cause of the EMS outbreak:

• The abstract for the first paper simply states that they traced the bad lot of L-tryptophan to a single manufacturer from Japan.
• The second abstract says the same thing (a different population was tested) and hypothesizes that a contaminant was introduced in the manufacturing process.
• The third abstract says the same thing, adding the point that the incidence of EMS decreased drastically once the product was recalled.

So far, there's nothing about a mysterious genetic engineering contaminant. There's reference to a contaminant, but no mention of when/where/how it could have been introduced.

If I were to magically design the perfect pomegranate, I think it would peel like a tangerine.

The next item I read was a 2001 report by the FDA (I thought it was odd that the IRT article did not mention the FDA's involvement). The FDA report was written up to clarify their position on tryptophan supplements. The report stated that 37 people died as a result of the bad batch of tryptophan. They state that there were numerous impurities in the batch, several of which were associated with EMS, but no one really knows how. They pointed out that not everyone who took the contaminated supplements got EMS, so there may be a genetic predisposition/factor involved as well. Again, nothing about a contaminant from the genetic engineering process.

I'd also magically make that white fluffy stuff disappear so that the seeds would just drop out. I looked it up and the white fluffy membrane doesn't have a specific name. You'd think it would have an uber-geeky scientific name.

Next, I found a New England Journal of Medicine article that looked into the whole tragedy. The article points out the fact that the manufacturer in Japan had switched to a new bacterial strain for the synthesis of tryptophan in 1988. In 1989, they made another change in their manufacturing process: instead of starting with 20kg of one of the starting materials, they cut it down to 10kg. During that same time period, some of the batches of tryptophan that they were making skipped one of their filtration steps. The authors find statistical significance between the amount of starting material used with EMS. They also find correlation between the bacterial strain used and EMS. However, they highlight numerous times that they cannot disassociate the bacterial strain used from the amount of starting material used, so they cannot tell the impact of the bacterial strain alone. Then they state: "For this reason, it is possible that strain differences were unrelated to the production of the etiologic [disease causing] agent." They also mention that the manufacturer's tests had shown no difference in the biological and physiological properties of the old and new bacterial strains. So the New England Journal of Medicine does not point to a weird by-product of the genetic engineering process. Also, article sheds a lot of light on the timeline: the first cases of EMS were identified in October 1989 and by early November 1989 the link between tryptophan and EMS had been found. It didn't take months or even years for this to be identified.

I only have two pomegranates left in the fruit basket... I'd better add 'pomegranates' to the shopping list. I wish I could write 'peel-able pomegranates'. I'd pay extra for those suckers.

So here's why I think that this is, by far, the most ridiculous "health impact" on the IRT's webpage and the molecular geneticist in me cringes at the thought that this is even considered a GMO, as defined in the GMO debate. In 1922, the first human patient received an injection of insulin isolated from a calf pancreas, and patients had to rely on insulin isolated from animals until the 1980's. To make human insulin, the DNA sequence of insulin is inserted either into bacteria or yeast, which then start making the protein. It is isolated, purified, and sold to millions of patients worldwide who rely on this lifesaving technology. Patients are not exposed to the bacteria or the yeast. These organisms are just used in the manufacturing process of insulin to make it scalable and, often, easier.

Statistically, insulin is a drug that me or someone I love and care for will probably end up taking in our lifetimes. And insulin isn't the only drug produced this way. Countless other drugs and dietary supplements,  including tryptophan, are produced using recombinant DNA technology (this article highlights that malarial drugs are being made this way). So why is the IRT focusing on this story about tryptophan and EMS? I really can't explain it.

In my perspective, the EMS outbreak highlights another issue altogether: the need to have more stringent regulations in the world of dietary supplements. The quick sequence of changes introduced in the manufacturing process of tryptophan and their immediate rollout to the market would have never even been contemplated if it were regulated as strictly as pharmaceuticals. Or if they wanted to make the changes, they would have had to perform rigorous testing to demonstrate that there's no change in the function of the product. Cutting back on starting material by 50%?? Changing the strain of bacteria used once the product is in the market? Utter craziness!! Please do not misconstrue this and think that I'm implying that the pharma/clinical world is perfect. Far from it. I worked on my first project in regulated markets this year and learned first-hand of the craziness that exists. But at least the regulations and trials that they have to go through put a few checks and balances in place.

Whew! With that rant I finish the review of the IRT's health risks. My conclusion: they focus on fear-mongering. Most statements were misleading or misrepresented the studies, and several statements were outright lies.

Till my next post, I'll continue enjoying pomegranate season and keep my fingers crossed that someone out there is working on a peel-able version of the fruit.

PS: After reviewing this, the spouse thought that the pomegranate seeds should be the size of grapes. Pure genius!