Gene Editing and GMOs

posted in: Science | 23

Gene editing has been getting a lot of attention lately, with an increasing number of articles about this method in the media. In this post, I’ll provide a very high level overview of the method (please note that many molecules and enzymes will be omitted for the sake of simplicity). Most of the information here is from a 2014 review entitled “Development and Applications of CRISPR-Cas9 for Genome Engineering” from the journal Cell (unfortunately behind a paywall).

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

The genome engineering methods currently used, particularly in mammalian cells, are difficult and inefficient. This has led scientists to search for programmable gene editing technologies, the most promising of which is the CRISPR-Cas9 system. Cas9 is an endonuclease, an enzyme that can cut both strands of DNA’s double helix. Some endonucleases are random, cutting 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 sequences appear multiple times in a genome, so cutting one specific location with a restriction enzyme is not an option.

Unlike restriction enzymes, bacterial Cas9 cuts at a specific site and 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 research.

There are different mechanisms by which the CRISPR system gets activated, and after many years of research, it was decided that CRISPR-Cas9 mechanism 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 the genome?

Once the DNA is cut, the cell’s natural repair mechanism kicks in and one of two things can happen (see the graphic below):

  1. The two loose ends of the DNA strand get glued back together again. This mechanism 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.
  2. 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 its sequence will get copied. The template that researchers provide can contain the desired sequence, additional sequence, etc. This process is known as homology directed repair.
CRISPR-Cas9 Gene Editing Mechanism. The CRISPR-Cas9 system cuts the DNA at specified sites in the genome. The cut ends are repaired via non-homologous end joining or homology directed repair. Image by Layla Katiraee.

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. For example, Cas9 can be fluorescently labelled/tagged, so researchers can visualize the location of the DNA sequence being studied.

In reviewing this article, my husband asked if we could write a movie script where the villain sprinkled Cas9 along with the DNA specific to the hero’s genome into the hero’s cereal. Would it be the perfect crime? Would the CRISPR-Cas9 enter the hero’s body and hack up his DNA? Unfortunately, no. Our DNA is within the nucleus of our cells and isn’t very accessible. Additionally, CRISPR-Cas9 exists only in bacteria. Getting the CRISPR-Cas9 system 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 (Miao et al 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.

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. 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?

The recent paper Regulatory uncertainty over genome editing provides a great summary of the regulatory issues. Huw Jones describes how 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.

Jones states that 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). Jones concludes two important points:

  1. If the EU’s definition of a GMO does not end up aligning with the USDA’s, 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, and stifle innovation. In other words, if you want someone other than Monsanto, Syngenta, et al to make a biotech crop, these crops should not be considered GMOs.

I think there’s much potential and promise in the system, and think it can be a valuable tool in the modification of crops just as transgenesis and mutagenesis have been in the past. I’ll keep my fingers crossed in hopes that gene editing might finally bring about my long cherished dream of a peelable pomegranate!

  • Giovanni Tagliabue

    Regarding the GMO regulation, the EU is embarrassed: some years ago, they asked a group of experts to assess whether using a group of “New Techniques” (“Oligonucleotide Directed Mutagenesis (ODM); Zinc Finger Nuclease Technology (ZFN) comprising ZFN-1, ZFN-2 and ZFN-3; Cisgenesis comprising Intragenesis; Grafting; Agro-infiltration; RNA-dependent DNA methylation (RdDM); Reverse breeding; Synthetic genomics”) creates GMOs or not, i.e. if the products deriving from the application of those techniques fall under the scope of the 2001/18 directive, with its contorted and incoherent “GMO” definition. A final report was written in April 2012 (almost 3 years ago!) and not published yet, because the scientists cannot give an impossible answer to a wrong question. The report has leaked though (www.infogm.org/IMG/doc/ue_working-group_nouvelles-techniques-modification-vivant_avril2012.doc, or http://www.seemneliit.ee/wp-content/uploads/2011/11/esa_12.0029.pdf) together with some comments from British and German scientific societies. I received confirmation from the EU: the report is authentic. The reason why it has not yet been published is clear: the “New Techniques” have already undermined the “GMO” definition, and CRISPR-Cas9 will create even more puzzlement. I’d like to hope that these advancements will persuade the EU legislator to rewrite the rules, focusing on products instead of processes, but I’m not confident at all…

    • http://frankenfoodfacts.blogspot.com Layla Katiraee

      That’s a very interesting document! It’ll be very interesting to see what the EU decides to do on this topic.

  • Ray Kinney

    No matter what food I eat, I want to know (to the best of my ability) just what I am putting into my body. GMO, non-GMO, organic… whatever, I want assurance (as best we can) that I know what is in the food… so that I can be basing my food habits as I see fit. That means, that I am all for GMO labeling… but that also means that that alone is NOT enough. Some system needs to be put in place for people to have far better knowledge than the current system practices. That means that our abilities at analytical assessment of chemical constituents that make up foods needs to become very much based upon pointedly investgative, ongoing, and pervasive monitoring. The problem seems to be that industry would oppose or obfuscate such knowledge base effort…why???

    • http://frankenfoodfacts.blogspot.com Layla Katiraee

      Hi Ray,
      Can you give us an example? Let’s say we’re looking at an apple pie made with apples, flour, butter, sugar. With the current labeling scheme, that’s all the information you’d get regarding the ingredients list. Let’s say that the apple is from a transgenic apple grown in BC, the flour was derived from a mutagenic strain grown in California, the butter was from Holstein cows fed GM grain in Vermont, and the sugar was from sugar cane imported from Ecuador. That’s the ingredient list for the apple pie batch made the week of March 3rd, 2015. What would you propose be the content of the ingredient list for the apple pie?

  • Ray Kinney

    Layla, sounds good, i would probably eat it if offered to me. and enjoy it. i would still be a bit concerned that none of the ingredients were organic. This of itself does not mean that it necessarily had any major red flag risks, but only that there could be risks included if chemical analysis were to be conducted to look more scientifically for potential contaminants, some of which might pose toxicologic risks to some people, especially to those that might be young, or older, or with medical conditions that make them less fit than the average pie eater. Also, if I were to eat this pie once or twice I might not give it another thought, but if I were goping to eat twenty such pies a decade I might want to know a bit more detail.

    • Chris Preston

      Ray, if you want organic ingredients there is already a way to ensure that. Ingredients carrying a label of organic have to come from enterprises that have been certified by one of the certifying bodies.

      What you will find is that no matter where the ingredients come from, they will contain chemicals of toxicological concern. Either because the plants (or microbes associated with plants or animals) produced those compounds or they were picked up during the growing, transport, production or packaging phases.

      So long as those chemicals remain at levels that are unlikely to have negative health implications, there is no need to be concerned.

  • Ray Kinney

    Layla, Now, it might be better, rather than testing such a pie, to propose testing the sources of the ingredients on an ongoing monitoring regimen, to look for known toxic substance content fluctuations over time, which might give better assurance of food quality. Systemic pesticide residues in the apples, or surface residues, or metals contaminants in the sugar, flour, butter, grain, etc. could be tested for. None of this testing need go onto a label unless it became known to be a significant problem as a contaminant in the food ingredient sources. If you were talking science, it would be good to know these foods were clean of these contaminants, if you were talking economics of profit margin, the average producer would probably want this analysis to be prevented. Organic producers on the other hand generally seem to want to know if there are contaminants that might be a problem in their products so that the food source could be cleaned up for the well being of the public (most but not all perhaps). Seems logical for food science to be done to standards such as these, as our knowledge increases, the screening process can evolve to better do the job. Currently, testing is far too insufficient IMHO.

  • Ray Kinney

    Layla, it would not cost very much at all to do such screening, especially when you take into consideration how, even a few problems would be discovered in time to prevent widespread, prolonged, exposures at health-relavent levels, might save society large sums of money and prevent huge medical system costs (and legal costs etc.). Seems logical to have such public health screening become a more robust science based food system. Pass the pie please!

  • Ray Kinney

    Chris,
    Your point is well taken.
    “So long as those chemicals remain at levels that are unlikely to have negative health implications, there is no need to be concerned.” However, that is precisely my concern. The science of toxicology is ‘ a science’ and as such, it is a dynamic changing, ongoing process of reevaluation as to toxicologic significance as new research is done. Research tools are constantly improving to provide us with better capabilities for evaluation at finer scales of knowledge. This means that we need to be fully aware that our onetime assessment of a particular point of food safety that was consistent with the then current knowledge base could always be subject to revision when new research suggests the need. The new science of epigenetics, the expanding sciences of low dose exposures at times being non-monotonic, and other significant advances in the science of toxicologic assessment often point to the need for reevaluation of the ‘safety’ assumptions of our past knowledge base.

  • Ray Kinney

    You are certainly correct in your point about all foods, organic and nonorganic etc. all having ingredients that could have toxicologic significance (both recognized and as yet unrecognized). My point is that we must stay up on all of the new science and make the food industry adapt to more effectively include the changing science in order to provide for better societal wellbeing.Often, there appears to be substantial, and even excessive, resistence to this adaptation impairative by the food industry… understandable from a more short term and more selfish corporate mandate to stockholder income rates and to ‘golden parachute’ style corporate manager sense of selfworth and ego that often tempts corporations to try to bias the science direction away from the pointedly investigative toward the profit generating staus quo position….. but this dynamic is my point…. and, I believe it also drives Vani Hari to do her ‘unscientific’ best to also provide for better food safety advancement in the face of the more egregious aspects of corporate irresponsibility. Hopefully, I will do what I do more gracefully in the future, hopefully so will she, and hopefully so will the corporations evolve toward even better food quality.

    • Chris Preston

      Ray, I agree that there is never complete certainty about toxicology, because some new piece of information may arise that changes our views. However, given the large number of chemicals, it is pretty rare for toxicological knowledge to be turned on its head. I think most people don’t understand epigenetics and use it as some sort of scare factor instead. Epigenetics is unlikely to overturn our understanding of toxicology.

      Toxicological studies are done in such a way that any adverse effect that impacts on the health of mammals can be identified. What we now have is the tools to measure a whole lot of changes that are not that important. People then make a song and dance about these unimportant details, which is unfortunate because they obscure the bigger picture.

      As for Hari, I am afraid I am a lot more cynical than you. She makes her money out of selling items from her on-line shop. Her articles are a way of getting customers. Scaring her potential customers into buying her products is an activity with a long and lucrative history in the alternative medicine industry.

      Her apparent complete ignorance of any chemistry is very unfortunate for someone who’s current operation is based on chemicals are scary. Or perhaps not.

    • http://frankenfoodfacts.blogspot.com Layla Katiraee

      Hi Ray,

      I agree that ongoing studies examining the safety of food additives, as well as pesticides, are necessary. I’d love to see the government agencies charged with the safety of our food to receive increased funding.
      Currently, the USDA tests conventional food for pesticides, to determine if levels of these chemicals are detected above the permissible levels (http://www.ams.usda.gov/AMSv1.0/getfile?dDocName=STELPRDC5049944). Of course, they do not test every supplier, but over the past few years, the trend has been the same: very few food items ever exceed the permissible levels of pesticide residue. Keep in mind that the permissible level is always set far below levels that are found to be harmful in animal studies.
      If I understand your argument correctly, your concern is over the fact that what we consider to be “permissible” today may not be the same value tomorrow, because we may discover a toxicological impact at a future point in time.

      I understand where you’re coming from. But if you start going down the slope of “we don’t know what we don’t know, so let’s test and label everything so that we can make informed choices”, where do you draw the line? We wash our clothes with approved soaps, we walk on carpets and hardwood floors that are treated with approved compounds, we type on keyboards and use electronics all of which use compounds and chemicals that have been approved. They all come into contact with our skin, emit compounds that we breath, and if you have toddlers, get licked and slobbered on a regular basis. Should all of these be regularly tested and labeled?

  • Ray Kinney

    Chris,
    I’d sure like to have confidence in the completeness and adequacy of the current state of toxicologic testing and assessment that you claim, but my readings of the research of toxicology of lead are extensive and I have consulted with a large number of researchers about their work and their confidence about the completeness of current knowledge…. and they have been overwhelmingly supportive of my understanding that there are still many areas of ignorance left to clarify that have very profound implications for societal harm ongoing. They are most insistent that epigenetics plays a big role, accumulative chronic low doses at environmentally significant levels, genetic differences in response, as yet unknown effects potentials on developmentally vulnerable life stages, elderly effects complications, etc. They are very concerned that behavioral effects that can be health-limiting are as yet far from being adequately defined, yet are still a major societal harm. Hundreds of these toxicologists are working hard to advance into these datagaps to advance our knowledge. My knowledge of pesticide effects research is far less extensive… yet still quite significant and indicative of less confidence.

    • Chris Preston

      Ray, the toxicology of lead is really quite well known. The safe exposure is below the current limit of detection, that is all exposure levels that we can measure likely lead to adverse measurable effects. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485653/

      What is problematic is ways to manage exposure. All the possible routes of exposure are not known and some of these routes are not able to be managed – e.g. lead in plants, lead exposure from soil and air.

  • Ray Kinney

    A thought about organic/ non organic food system safety craziness. Both types of food, if intended by the industry to be ‘state of the art for safety’ and public wellbeing, still do very questionable practices such as putting labels proudly onto their finished products with BPA-laced labels. Organic foods with such label contamination are crazy… any food product with such label seems less than intelligent IMHO. Hopefully there is current movement away from this problem.

    • Rickinreallife

      You brought up BPA which is an opportunity to also point out selectivity in risk and how obsession with one type of risk, theoretical or well defined can skew or overshadow other known risks. . I know that there is concern about BPA in plastic resins that are used to line food container, i.e tin cans. I am not even dismissing that there could legitimate basis underlying that concern. I am only using numbers made up to illustrate my point, and I don’t have actual data at my fingertips as I type this. But what if you were to learn that since the use of such resins, the number of cases of botulism traced to canned food products dropped from 100 cases annually to say 5 cases annually, related deaths from a couple a year to 1 every 5 years. We know that some foods that come into contact with food container linings, say canned organic tomatoes, are caustic and can dissolve small amounts of the tin without the plastic resins. What if tomatoes packaged in a non resin lined can for 6 mos had say 5 parts per million tin residues and those in resin lined cans had only 1 pert per million. To save ourselves from miniscule exposure to BPA, we subject ourselves to 5 times the amount of heavy metals.

      Rejecting BPA containing resins that may be a food contact substance does not solve the problems of protecting the public from botulism and heavy metal exposure, it just rejects one solution to that problem. There are alternatives to BPA containing resins as can liners, but these are inferior to BPA resins in many important ways –they may degrade more quickly, exposing consumers to higher amounts of other types of residues that might be more harmfule than the miniscule exposure to BPA. They may not reteain adhesion over time as well due to temperature extremes, jostling, disfiguration of the can, etc. Some alternatives are not universal, they might degrade in contact with acidic foods like tomatoes.

      That is why I am skeptical about required labeling of BPA resin lined containers — I actually think it would be a net loss in food safety, and that labeling disclosure fails to warn of the heightened risks of non-BPA alternatives that might be used. A required label disclosure for BPA doess not give consumers a full, fair and accurate set of information to make an informed choice in their self interest.

      A lot of the risks we associate with genetic engineering, BPA, food additives, etc. are a lot like if you are watching a football game, and you only have refs looking for and calling penalties on one team. One team appears to be violating the rules of play regularly, holding, offsides. Its not that the otherteam didn’t also commit those penalties, its just that there was nobody looking for penalties.

      I think FDA has called this one right on BPA. I am not saying that BPA exposure could not conceivably entail some risk. I am not even opposed to the idea of continuing to look for alternatives that serve the many food safety functions tha BPA containing resins have but avoids the risk of BPA leaching. But in my opinion, the provable and theoretical risks due to the minimal exposure through diet are far outweighed by the known risks of less effective food container alternatives.

  • Ray Kinney

    Hi Layla,
    We would probably all like to see better monitoring of food safety as we are able. My point is that we know a lot about math, physics, and chemistry, but, the science of physiologic complexity of biochemistry is still far less defined, more subject to discovery of mechanisms of potential adverse biochemical effects that we have not taken adequately into account as yet. For example, the new science of nano technology is going to very rapidly disperse huge numbers and quantities of products out around the world, likely, many will enter the realm of food production. We do not know very well just what toxicologic adverse effects potentials will come out of these introductions because of all the datagaps and research yet to be done for reasonable due diligence. The very small size of the particulates allow for many physiologic behaviors to differ from those we are more used to and have studied more thoroughly. Just one example, that together with the newer understandings of epigenetics brings a lot of pressure and uncertainty to the toxicologic potentials within the food safety paradigm as well as elsewhere.

  • Ray Kinney

    I guess, I’m just saying that we should raise the bar higher as we are able. Of course, we will not be able to test everything. The ‘line’ should not be drawn, but be increasing our knowledge in an ongoing fashion driven by our desire for the wellbeing of our great grandchildren and their children. I firmly believe that we can greatly improve the health of most people, saving a great deal of money and physical ailments, by further clarifying existing toxic effects in foods, and adjusting our best practices for production and delivery to take advantage of what we learn to promote even safer food and health.

  • Ray Kinney

    Chris, Yes, the toxicology of lead is extensive, yet there are so many biologic pathways affected that large areas of adverse effects have very important datagaps to be clarified, especially behavioral toxicology. we are only seeing the ‘tip of the iceberg’… and, we are still weighting our fishing lines with lead sinkers that poison us, our families, and the fish we handle. It is a VERY toxic contaminant that we still don’t manage exposures to well.
    All of this should be said about the state of the art of toxicology of many AG use pesticides as well, especially ones slated for incorporation in staple crops as pesticide resistent GMOs (glyphosate, diazinon, 2,4D) since the exposures are to become such vast sociologically significant risks if some health-limiting aspects have been understudied to date. Apparently you assume that all the bases have already been covered and ‘done deal’.
    Yes, I am skeptical! We may think we know a lot… but there are certainly many risks lurking unseen yet to discover. The real world is messy, and so is the science of ecotoxicology of mteals.. and of pesticide unintended consequences… soon our understanding will be seen as still being very primitive and harmful because we thought we knew enough to widely disperse these products throughout our food supplies.

  • Ray Kinney

    “NEW STUDY: Goats fed Monsanto Roundup Ready soy produce abnormal milk. GM-fed goats milk has significantly reduced antibody, fat and protein content and also co…ntained transgenic DNA. Colostrum, the nutrient-rich first milk of nursing mothers, is ordinarily packed with proteins, vitamins as well as antibodies to protect the new-born against disease. Offspring from conventional and GM-fed mothers weigh the same at birth but those from GM-fed mothers weigh significantly less 30 days after birth. This study provides further evidence that GMO soy is not substantially equivalent to NON-GMO soy, with clear negative impacts. “The US has by far the highest level of neonatal mortality in industrialized nations. Many factors likely underlie this statistic but it is clear that the integrity of the first meals of a baby’s life has far-reaching and long-term effects.”
    DOI: http://dx.doi.org/10.1016/j.smallrumres.2015.01.023
    Well, what’s to it folks? More bad science, or something to consider?

  • Ray Kinney

    Rick, Re: BPA/metals container issues…. The real point is that perhaps we could avoid all of those toxicants by using glass containers rather than cans. The food producers would howl, but the costs would really just be put back onto the public anyway, however the costs of the toxicant effects of canned food use that bring chronic low accumulative effects on human pathogenicity cost society untold funds, lost human potential, and pain… that is the real world. If can’t figure out how to can food well (in cans) without poisoning people, use glass.The money saved could be used to do environmental monitoring of other such food supply contaminant risks with the intent of finding alternative to save even more money and human siffering.

  • Ray Kinney

    Chris, your quote: “What is problematic is ways to manage exposure. All the possible routes of exposure are not known and some of these routes are not able to be managed – e.g. lead in plants, lead exposure from soil and air.” is certainly correct.. and applies to both lead toxicology and to pesticide resistant crop production. And, industry found a loophole in regulations years ago that showed them how to reclassify their toxic waste from smokestacks (that they had to pay for to dispose), and allowed them to call this toxic waste ‘soil ammendment’, thus contaminating vast amounts of AG land… polluting often pays… the Manchester lab finally discovered the problem and EPA closed the loophole… now, we just have to continue to pay as we eat the resultant contaminants in the food supply. Society pays over and over again for short term industrial irresponsibility of externalizing costs to produce false profit. Happens all the time, the system needs a lot of improvement to becom socially responsible.

  • Ray Kinney

    Pass the pie please, errr the organic pie please.