Better Know a Scientist: Interview with Estefania Elorriaga on Site-Specific Nucleases

This week in “Better Know a Scientist”, I’m interviewing Estefania Elorriaga. She’s in the midst of her PhD in Dr Steven Strauss’ lab in the Department of Forest Ecosystems and Society at Oregon State University. She is doing research on using site-specific nucleases for mutagenesis (fear not! She’ll have to explain her research in this interview). Let’s get started!

Q: What are site-specific nucleases? Why are they important?

Site-specific nucleases are enzymes that can cut DNA at specific  locations in the genome of your organism.  The nucleases create a break which stimulates the organism’s DNA repair mechanisms to fix the break.  Occasionally, the repair mechanisms will make a mistake (delete some DNA or insert some extra DNA) that will lead to a loss-of-function mutation in the target gene, meaning that the protein you targeted will no longer do its job.  Examples of site-specific nucleases are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system, and engineered meganucleases.  Site-specific nucleases can also be used for gene repair or gene replacement with the addition of a donor DNA sequence.  These technologies are important because they allow scientists to modify only specific locations of genes in order to obtain a desired trait.  In the area of crop breeding we believe these technologies will be crucial for improving current lines specially for particular cases like drought-tolerance, high or low temperature tolerance, disease resistance, or even nutrient content.

[Biochica’s note: if you want to learn more about gene editing, please see my previous post on the topic]

Estefania's work using TALENs.
The plant on the left has a TALEN (will show it's trait much later)
The plant on the top-right is her transgenic control,
expressing GFP (green fluorescent protein).
The plant on the bottom right is the unmodified control.

Q: How specific are the nucleases? If you can share, are you seeing off-target edits in your work?

We are still at the early analysis stage, so I cannot really say personally how specific they are.  But, according to the scientific literature, CRISPR-Cas nucleases appear to be the most active and specific, followed by TALENs, and last ZFNs.  CRISPR-Cas nuclease show a lot of promise because of the high activity that they show so far and their off-targeting appears to be somewhat predictable.  

Q: Are there any GMOs made using site-specific nucleases currently on the market? Do you know of any that are in development?

There are no commercially available GMOs currently in the market or in development (that I know of), but everyone in the field believes that the technology is going to revolutionize genetic engineering, especially breeding and gene therapy.

Q: Like me, you were raised in Venezuela (I was raised in Barquisimeto and I left after graduating from high school). Venezuela has a moratorium on growing any GMOs and activists would argue that the moratorium is in place because of how harmful GMOs can be. So, how do we know that you aren’t a spy sent to the United States to bring down the imperialist empire through evil GMOs?

Ha ha! If the Venezuelan government was making me get a PhD just to spy on the USA, I think I would have quit my job as a spy and gone to Colombia or Costa Rica.  I cannot imagine doing research while spying.  As it stands, I barely have any free time.  And also, I cannot imagine pretending to be a researcher.

[Biochica's note: that's exactly what a person tasked with destroying the imperialist empire with GMOs would do: deny it].

Q: What traits are you working on in the lab? Why are they important?

I work with flowering genes in both poplar and eucalypts. Understanding floral development and identity genes is important because it will allow us to generate transgenic trees that don’t have functional pollen or ovules, so there is no possibility of transgene flow.  Developing transgene containment technologies for forest trees to facilitate the commercial and scientific use of transgenic trees is one of the main goals of our lab.

Q: Can you explain what you mean by “transgene flow”? [Biochica note: some activists refer to this as “GMO contamination”] Does it basically mean that you’re blocking the GM tree or plant from hybridizing or crossing with a non-GM tree or plant?

“Transgene flow” is the movement of the gene we inserted into our trees into a wild or cultivated population of the same or a related species. And yes, it basically means not allowing our GM tree to cross or hybridise with wild or cultivated trees.

Q: If you’re creating a GMO to address one of the major concerns against GMOs, aren’t you creating a circular argument? How would that conversation even go? I imagine it would be like, “Activists! You’re worried about GMO genes getting into the environment? Here’s a GMO that will prevent GM genes from getting into the environment. Go forth and plant it!” Aren’t a few heads going to explode? Is THAT the real plan of the Venezuelan government? To bring down the imperialist empire through circular arguments?

By eliminating transgene flow, we are just allowing the GM trees to be used commercially for other purposes like a non-flowering faster growing eucalyptus.  Also, eliminating transgene flow in the current GM crops will allow the scientific community to educate the public, and also other scientists that are weary, about the safety of GM technology.  We hypothesize that transgenes will likely not do well in the wild, so they will probably get eliminated through fitness selection, but we need to study each particular case. Ha ha! Nothing from the current Venezuelan government surprises me, so who knows...

Q: Are there any non-GM crops where gene-flow is a problem that may benefit from the adoption of the technology?

The other case that comes to my mind is the case of gene purity in seed crops.  Seed producers follow standards and practices that guarantee the purity of the seeds available to farmers and home growers. Genetic purity in their case is essential to ensure that the seed will perform as expected, so seed producers must be careful about the pollen that fertilizes their plants.  

Q: What trait would you like to work on in the future?

I would like to work with either nutrients (e.g. create a highly nutritious fruit or vegetable crop), drugs (e.g. create plants that can make medicines), or abiotic resistance (e.g. create plants that can take up heavy metals).

Q: There are many articles in the news and in journals about potential traits that may benefit us, many of which never make it past research, but none-the-less create much buzz. Which one(s) do you think are the most exciting? Which one(s) do you think are the most promising?

Being a transplant in Oregon and feeling like a true Oregonian since I moved to the Northwest, protecting the environment is a topic dear to my heart.  In the University of Washington, Prof. Sharon Doty worked with transgenic poplars that were able to remove more than 90% of contaminants from Superfund sites. According to the EPA, “A Superfund site is an uncontrolled or abandoned place where hazardous waste is located, possibly affecting local ecosystems or people”. I think using trees to clean our aquifers and our soils is a win-win. We get more oxygen, cleaner air, and also cleaner soils and rivers. But, the trees are nowhere near being ready for use outside of university labs.  

There is also a transgenic pig called “the Enviropig” from the University of Guelph in Canada, that digests phosphorus from its food more efficiently than non-transgenic pigs, so it needs less feed and its waste is less toxic to the environment.  Doesn’t that sound like a win-win too?!  

I am also a big fan of producing medicines in plants (like I mentioned above).  This practice is known as biopharming.  I am also a fan of using GM mosquitoes to eliminate vector-borne diseases like dengue (after the GM mosquito mates with a non-GM mosquito the transgene causes the mosquito larvae to die).  These two cases I mentioned though are closer to production than the Enviropig or phytoremediating trees.  Biopharming had a rough time recently because many of the companies involved went bankrupt, but there is an experimental antibody for Ebola currently being made in tobacco by Kentucky BioProcessing called ZMapp that should give this industry a boost, and the British company Oxitec plans to release GM mosquitoes in the Florida keys to reduce dengue and chikungunya (both diseases you don’t want to get, and less yet while on vacation).  ZMapp is considered the most promising candidate to combat Ebola and it is currently being used in a controlled human trial in Liberia. The mosquitoes were already tested in Brazil and Panama with great success, so both countries plan on releasing a lot more of them.

[Biochica’s note: if you want to learn more about Oxitec’s mosquitoes, please see my previous post on my family’s experience with dengue and my review of the literature on these phenomenal skeeters]

Q: How much money is Monsanto paying you to develop these transgene containment technologies that will solve one of their public image problems? After all, we all know that Monsanto controls university research.

Monsanto has never paid me or our lab for the research we do.  Sadly, when many people think “GM technology” they think “Monsanto”.  But, in reality the technology is being used by hundreds of university labs, research facilities, and biotech companies developing more than herbicide- or insect-resistant crops.  Monsanto and all the other large biotech multinationals like Dupont, Pioneer, or Syngenta come up with cutting-edge seed and agro-chemical technologies, but given that there is no direct benefit to the end user (all the benefits go to the farmer), the public shows little to no support because of lack of understanding and fear.  If you look over the ISAAA’s (International Service for the Acquisition of Agri-biotech Application) Pocket K. Documented Benefits of GM Crops, you will find that from 1996 to 2012, global farmers’ income increased by $116.6 million billion and there was a reduction in herbicide and pesticide use of 503 million kgs (ISAAA is an international non-profit “that shares the benefits of crop biotechnology to various stakeholders, particularly resource-poor farmers in developing countries, through knowledge sharing initiatives and the transfer and delivery of proprietary biotechnology applications”).  All these agricultural biotech companies perform cutting edge science and create impressive biotech products.  However, as companies they are concerned with profits, so given the high cost of generating a GM product, they focus on products that will bring them a return on their investment.

Q: My dreams for crop modification aren’t as lofty: I just want a peelable pomegranate. Do you think that the research you’re conducting might be able to help?

My work won’t directly affect the possibility of creating a more peelable pomegranate. But, indirectly it can bring science closer to your dream.  My research can add onto the increasing scientific knowledge base that is proving that site-specific nucleases can someday be an important tool in crop breeding.  If we find the gene or sets of genes involved in making the pomegranate’s skin, we can either modify or replace the genes for ones that will make the pomegranate easier to peel.  For many centuries, humans have been modifying the aspect, size, and taste of cultivated crops by doing selective breeding without knowing anything about genetics.  Wild bananas and corn (and dogs...chihuahuas come from wolves!) are great examples of what selective breeding can do.  Wild bananas are small and loaded with seeds.  Meanwhile, cultivated bananas are large and have tiny seeds (that actually are not viable because cultivated bananas are sterile).  Teosinte (the wild ancestor of corn) make small ears with only two rows of hard fruit cases that protect the seeds.  Corn makes large ears with eight to twelve rows of tender seeds (no hard fruit cases).  So, GE is basically streamlining breeding by allowing use scientists and plant breeders to perform highly selective and direct breeding using genes from the same species or other species.

[Biochica’s note to the spouse: it sounds like there’s the remote possibility that Estefania’s research might be able to make my pomegranate. Please be advised that we may have to move to Oregon in the near future to help conduct this research].

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.