Precise Genome Engineering

For many years genetic engineering of plants has been limited to inserting a DNA sequence of interest at a random location in the genome. It was not possible to choose where in the genome the transgene integrated. Furthermore, it was not possible to delete or modify existing genes in the plant genome. Recently several technologies have emerged that allow for making insertions, deletions, or modifications to a plant genome at desired locations.

Creation of double-stranded DNA breaks

Technologies to make precise changes to the genome rely on the ability to make double-strand breaks in the DNA at specific locations. There are several methods by which to do this including the CRISPR/CAS9 system, TALENs, and zinc finger nucleases. The CRISPR system is the newest one, first published in 2012, and is the easiest to direct against a desired location in a DNA sequence. Once present, the double strand break is repaired by endogenous DNA repair pathways that exists for repairing naturally occurring double-stranded breaks. The way in which the DNA repair occurs dictates the type of modification made to the genome

Disrupting a specific gene by introducing a mutation

One of the DNA repair pathways, called non-homologous end-joining or NHEJ, joins two pieces of double stranded DNA without using a repair template. This process is error-prone and often results in the deletion or insertion of several DNA base pairs. If located within a gene, such mutations are likely to disrupt the function of that gene. Scientists can therefore use this method to disrupt to function of a particular gene, either for research or commercial purposes.

Precise editing of a gene

An alternative DNA repair pathway present in plants (and many other organisms) is called homologous DNA repair. As suggested in the name, this type of repair uses a DNA with a similar sequence to the broken strand to make the repair. Under ordinary circumstances the repair template may be the DNA from the sister chromosome. Scientists can supply an alternative repair template containing borders that match the regions flanking the cut site. Once repaired, the targeted region of the genome will match the supplied template DNA. Based on the design of the repair template and the cut site or sites, it is possible to make changes ranging from the alteration of a single base pair to the deletion of many contiguous genes.

Getting the genome editing components into the plant

The machinery to make a targetted double strand DNA break along with a DNA repair template is can be delivery by traditional methods for making transgenetic plants (typically through the use of Agrobacterium or particle bombardment). These components will typically integrate into the plant genome prior to being expressed and eventually leading to the desired genome modification.

Removing the genome editing components

As the genome modification is permanent, the machinery for making the double-stranded DNA break is not required once the modification has occurred. For research purposes having these components would likely not be a concern, however for commercial use it may be desirable to remove these components from the genome. This can be done quite easily by crossing the plant and selecting progeny that contain the desired genome modification but have segregated away the editing machinery. Alternatives exist to remove the construct from plants that cannot be easily crossed, though they would likely result is a small "foot print" of exogenous base pairs left behind.

The final product

While traditional genetically modified crops contain transgenes located at an arbitrary location in the genome, plants that are the result of precise genomic engineering could be genetically indistinguishable from plants obtained through conventional breeding or spontaneous mutations. For instance, these methods could be used to insert, delete, or change only a single base pair in a gene. Alternatively, an allele for a trait, such as disease resistance, from a wild variety of a species could be introduced at the orthologous position in the crop variety. This would be much faster than traditional breeding and would eliminate potential risks from linkage drag of undesired genetic material. These types of engineered crops, not actually containing foreign DNA and very much resembling alterations that occur through natural processes and traditional breeding, pose fewer risks and are likely to face less regulation than conventional GM crops.

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