Regulatory issues concerning CRISPR/Cas9-modified plants

The efficacy of CRISPR/Cas 9 technique to obtain precise genetic modifications makes it more difficult to identify a genetically modified plant one outside the lab and also to regulate this organism in the market. For a consumer and also a regulatory agency it would become nearly impossible to find out which organism was genetically modified and which does not. (Ledford, 2015) Furthermore, for many years modified organisms have been patented when they had an industrial use. With the new CRISPR/Cas 9 a regulation of the patenting is almost impossible due to many economic interests that are involved and the modification is hard to prove. (Rodriguez, 2016)

Although Switzerland has one of the most restrictiv laws concerning genetically modified organisms the current regulation in Switzerland does not cover the use of the CRISPR/Cas 9 sufficiently. Before a genetically modified organism can be put into circulation it must be researched and the confederation needs to grant authorisation. (BAFU, 2004) In my opinion, the biggest issue in the Swiss laws is the definition of a genetically modified organism: “Genetically modified organism means organisms in which the genetic material has been altered in a way that doesn’t occur under natural conditions by crossing or natural recombination” (Gene Technology Act, Art. 5, BAFU).

For that reason, organisms which have been modified with the CRISPR/Cas 9 system do not fall under that articles of law and would not be called GMOs according to the Swiss regulation. But how would you describe and regulate CRISPR/Cas9-modified organisms when the modification is hard to prove? I personally could not answer that question and time will show if anyone can.

If I had to create a forecast what the Swiss public would say about the use of CRISPR/Cas9-modified plants, the Swiss citizen would definitely reject a use of such a plant on the market. From my point of view, ignorance and fundamental miscommunication by the media would be the main problem. People are always afraid of new technologies and not without a reason. History has shown mankind many times that late results can always arrive and we as human beings do not learn anything from it. Even though the CRISPR/Cas 9 system is such a powerful tool, time is needed for a wide spread social acceptance.

References

Federal Office of Environment (BAFU) (2004) Federal Act of 21 March 2003 on Non-Human Gene Technology (Gene Technology Act, GTA) Retrieved on 7.5.2017 from https://www.admin.ch/opc/en/classified-compilation/19996136/index.html

Ledford, H. (2015) CRISPR, the disruptor. Nature 522: 20-24 found in Rodriguez, E. (2016) Ethical Issues inn Genome using Crispr/Cas9 System. J Clin Res Bioeth 7: 266

Rodriguez, E. (2016) Ethical Issues inn Genome using Crispr/Cas9 System. J Clin Res Bioeth 7: 266

CRISPR/Cas9

The CRISPR/Cas9 method is based on a natural system used by bacteria to protect themselves from infection by viruses. When the virus inject its DNA into the bacteria’s cell enzymes cut a piece of the viral DNA out of its helix and integrate that into its own DNA on a specific site called CRISPR sequence. The cell will copy that sequence into a crRNA and a tRNA. These two RNA’s bind than onto a protein and form a complex called CAS9. CAS9 is an endonuclease, a type of Enzyme that can cut DNA. When the matching sequence known as a guide RNA finds its target in the viral Genome the CAS9 will start to unzip the DNA and match it to its target RNA. If the match is completed the CAS9 will use two tiny molecular scissors to cut the DNA. When this happens the cell try to repair the cut but the repair process is irreversible leading to mutations that can disable the gen. Over the past few year researcher studied the system and realize that it could be engineered not to just cut viral DNA but any DNA sequence at a precisely chosen location by changing the guide RNA to match the target. To be even more precise a DNA can be added which carries the desire sequence. Once the CAS9 made a cut the new DNA template can pair up with the cut ends, recombining and replacing the original sequence with a new version.

In wheat, pow­dery mildew is one of the most destructive plant pathogens worldwide. Bread wheat is an allohexaploid, with three similar but not identical copies of most of its genes. For this reason, breeders have not managed to breed a mildew resistant plants to this day. But the CRISPR/Cas9 system could lead searcher to revolutionary results. By targeting the three called MLO sequences, which encode proteins that were shown to repress defences against powdery mildew diseases in other plants, a durable resistance to the fungal pathogens that cause powdery mildew could be created. (Wang et al., 2014)

In order to achieve the desired genome editing in the target cell the Cas9 protein and the sgRNA needs to be delivered into the nucleus of the living cell. Those two key elements of the CRISPR/Cas9 system can be can be expressed either from expression vectors or by microinjected RNA and mRNA. By using the CRISPR/Cas9 approach the most common delivery method in genome editing are the Agrobacterium-mediated transformation, the polyethylene glycol-mediated transformation and the shotgun method. Furthermore, the selection of the matching promoter and a codon-optimized versions of Cas9 are very important for efficient genome editing. (Kumar and Jain, 2015)

Discussing the advantage and disadvantage of new barley known technology is a challenging task. Fact is the CRISPR/Cas9 system is a very powerful tool in genome editing. But as it is the case with any new technology, the benefits of CRISPR/Cas9 are followed by equally huge risks from potential misuse, but also unforeseen consequences. Since the DNA of a CRISPR-modified plant doesn’t interfere with a control group, I don’t believe that the consumption of such a plant is dangerous. About what I am worried about is potential loss of biodiversity and the missing regulation of the government worldwide which causes possible misuse.

Refrences

Kumar, V. and Jain, M. (2015). The CRISPR–Cas9 system for plant genome editing: advances and opportunities. Journal of Experimental Botany Vol. 66: 47–57

Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C. and Qiu J.-L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology 32:1-6

Micropropagation of Morus alba var. Shidareguwa

What did the researchers consider when inoculating Morus alba?

A crucial part of this research was stage 0 of in vitro culture which dealt which plant selection and disinfection. For that reason, only healthy, young growing shoot containing tips and nodal segments were randomly collected from 3-year-old field-grown ‘Shidareguwa’ trees. In order to create optimal growth conditions the nutrient composition of the media and the chemicals used to sterilize the plantlets were tested in pre experiments. (Aroonpong P., Chang J., 2015)

Which measures are available for shoot induction and rooting in vitro?

The induction of shoots and roots can be controlled by the selection of Medias with different concentrations of phytohormones and nutrients. For an optimal growth shoots and roots dependent on an amount of 3% of sucrose (Sajeevan et al., 2011) and 0,8% agar in the media and adjusted to pH 5.8. (Aroonpong P., Chang J., 2015)

Which conclusions do you make when reading Figure 2 of the publication?

In Figure 2 clearly shows a direct correlation between the Survival rate and endophytic contamination of the explants. The reason therefore leads to the meristem of the shoot tips which aren’t contaminated with endophytic bacteria yet. Node 3 shows a higher contamination rate as its closer to the media which is the main food source for fungi and bacteria. In my opinion the increased browning on nodes 1 and 2 could indicate that they are weaker compared to the shoot tip and node 3 and therefore more often targeted by pathogens. (Aroonpong P., Chang J., 2015)

Which factors are crucial for the last step of in vitro culture: acclimation?

An important factor of stage 4 of in vitro culture is the gently acclimation of the plantlets to their new surroundings. Therefore, the young plants are covered with a plastic tube and in order to adapt to different conditions. Furthermore, an optimal temperature in the green-house and a well grown root system is crucial to avoid a shock in their metabolism. (Aroonpong P., Chang J., 2015)

Refrences

Aroonpong, P., Chang J., 2015, Micropropagation of a difficult-to-root weeping mulberry (Morus alba var. Shidareguwa): A popular variety for ornamental purposes. Scientia Horticulturae 194 (2015), 320–326.

Sajeevan, R.S., Singh, S.J., Nataraja, K.N., Shivanna, M.B., 2011. An efficient in vitroprotocol for multiple in mulberry, Morus alba L. variety V1. Inter. Res. J. PlantSci. 2, 254–261.