Genetic modification with a twist!

DNA editing has always been a controversial topic, with many countries around the globe, particularly in Europe, refusing to grow or import crops that have been genetically modified. General concerns range from risk of cross contamination of GM crops with non-GM plant species, to risks to human consumption of GM foods.

Many experiments have been run to test different outcomes and effects of genetically modified crops, to identify advantages and any disadvantages or risks to cultivating GM on a commercial scale. Several countries already grow a considerable amount of GM crops, though most of these crops are for animal feed or for use as biofuel, with the US, Argentina and Brazil as the biggest growers of GM.

Because of the concerns about GM, scientists are now trying to find other ways to manipulate plants in order to improve crop production. And we might have a possible solution.

A team in South Korea, led by Je Wook Woo at the IBS Center for Genome Engineering in South Korea, have recently published a paper, outlining their most recent experiment, where they managed to edit and manipulate plants without inserting foreign DNA into the genetic code. As it stands, conventional genetic modification results from inserting DNA from another species of plant or other organism into the chosen plant for modification.

Here’s some background information. Genome editing has actually been used for quite a number of years, used to enhance many beneficial qualities in crop plants. It creates very precise targeted mutations within the cell and uses artificial enzymes – molecules which create breaks in the DNA sequence. Under natural circumstances, if a break occurs in the DNA sequence within a cell, the cell will automatically try to repair it using one of two repair mechanisms, either non-homologous end joining or homologous recombination.

DSBs

A single stranded break, or double stranded breaks can create mutations in DNA.

Non-homologous end joining basically involves joining the broken DNA strands together, and is really susceptible to errors, often mutating and inactivating a gene as a result. However, if the cell has a suitable DNA template available for repairing the broken DNA strands, it will recreate the DNA sequence that was lost or broken via homologous recombination. This process of homologous recombination is actually more accurate, so you get an exact copy of the original DNA sequence, and everything is as good as new.

Scientists can artificially make breaks in the DNA sequence of a plant using an artificial enzyme. Instead of allowing the cell to provide its own repair template, scientists can provide a synthetic repair template. The synthetic repair template could be a new DNA sequence which codes for a new gene, for example giving the plant resistance to a particular disease. One genome editing technique used is the CRISPER-Cas9 system.

This system was first identified in bacteria, as part of their immune system response if under pathogen attack, and scientists thought it would be a good idea to use it for targeting genes for genetic modification. The CRISPR-Cas9 system uses a molecule, called a single guide RNA, to find and latch onto a gene which is the target for genetic modification. An enzyme, called Cas9, comes along and breaks up the targeted gene. The cell then repairs the broken DNA sequence using a synthetic repair template. And voilà you have a new gene incorporated into your plant’s DNA.

What Je Wook Woo and his team did was create pre-assembled ribonucleoproteins, which are effectively protein-RNA complexes. They mixed the Cas9 enzyme with single guide RNA molecules which targeted genes from 3 plant species. Once they had this mixture of molecules, they infected plant cells from tobacco, lettuce and rice with it to create mutations in the target genes.

Nature article Je Wook Woo

Lettuce seedlings with the edited DNA sequence (taken from Je Wook Woo’s paper, see link below).

The results were that the Cas9-ribonucleoprotein complex created mutations in the target genes of the plant cells immediately after infection. The Cas9-ribonucleoprotein complex was quickly degraded by the cell once it had created the mutations so it couldn’t mutate anything else in the cell. The bonus factor of this experiment, was that these gene mutations remained in the DNA even after the plant cells had regenerated themselves. The team was able to grow seeds from the plants with the edited DNA, and the offspring still maintained the genetic mutation.

nbt.3389-SF5

Different stages of tobacco plant cell regeneration, showing wild type (WT) and mutant (bi-allelic KO) plants (taken from Je Wook Woo’s paper, see link below).

No need for inserting foreign DNA as a repair template!

The IBS team have made a revolutionary discovery; they have now paved a path to engineer plants that might not be subjected to GMO regulations, since foreign DNA was not actually inserted. Hopefully, in the next few years, we can see some more progress in this area.

Saving agriculture one cell at a time!

Link to journal paper:

Woo, J. W., Kim, J., Kwon, S. I., Corvalan, C., Cho, S. W., Kim, H., Kim, S.-G., Kim, S.-T., Choe, S. & Kim, J.-S. (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nature Biotechnologyhttp://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3389.html