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):
- 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.
- 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 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:
- 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.
- 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!