Regulatory issues concerning CRISPR/Cas9-modified plants

Overview

From the very beginning, genetically modified organisms (GMO) and their use have been subject of countless discussions. Even today there isn’t a public consent, whether we should use all the possible methods to modify and improve certain species or better keep our hands off it. The CRISPR/Cas9 technique and its ability to knock-out or insert specific genes has shown its potential in a various amount of different research topics. At the same time more and more people are afraid that scientists don’t know their limits and the discourse about the ethical and economic guidelines are years behind. Worldwide regulations are non-existent and therefore every country has its own vision what ethically is fine and what not. The question now is, what kind of regulations are already existent in Switzerland and what is the estimated public opinion on the subject?

Regulatory issues

In Switzerland a genetically modified organism is defined as:

“… an organism, which genetic material was manipulated in a way that could not be possible with crossbreeding or natural recombination.” (Der Schweizerische Bundesrat, Gentechnikgesetz)

For example, in wheat it’s extremely difficult to knock-out certain genes with cross breeding or with radiation because the chromosomes are hexaploid and the gene is therefore six times present. (GenSuisse, o. J.)

CRISPR/Cas9 made this step faster, easier to do and a lot cheaper. Following the Swiss definition of GMOs, this method is showing that it can produce manipulated plants at a time rate which are almost impossible to achieve in a natural way. Conclusion: Plants which were modified with CRISPR/Cas9 are GMOs even though the plants can’t be differentiated from types which were bred with conservative methods. (Lahrtz, 2018)

Figure 1: Overview of the different modification types and the regulatory issues.
Picture from the NZZ article (see references below)

In 2018 the European Court of Justice decided that plants which were manipulated with the CRISPR/Cas9 technique must be labelled as GMO and need to fulfill all the regulatory requirements which are attached to it before they can be used in the open. (Michael Lange, 2018)
It’s likely that the Swiss authorities will follow the same argumentation.

Additionally, issues with mutations in unintended targets (known as off-targets) have yet to be extensively investigated across plant species. (Lahrtz, 2018)

Conclusion

To put my personal opinion into it. It’s clear that plants which were modified with CRISPR/Cas9 need to be declared as GMOs. The thing is. People still tend to believe that products which are GMOs are in some way not safe, or not healthy, even though there is still no evidence for it. Therefore, a lot of products won’t have a chance on the market when they must be labelled as genetically modified. This is in many ways linked with side effects. As an example: Farmers won’t use seeds from genetically modified plants because they know that the consumers don’t want them. That means, they will still have to use crops that are in many ways less adapted to new biotic or abiotic conditions compared to GMOs. Higher water usage and a higher demand for pesticides is the logical consequence. This can’t be in anybody’s interest. Not the farmers and surely not the consumers. 

References

Der Schweizerische Bundesrat. (o. J.). SR 814.91 Bundesgesetz vom 21. März 2003 über die Gentechnik im Ausserhumanbereich (Gentechnikgesetz, GTG). 

Zugriff am 7.5.2019. 

Verfügbar unter: https://www.admin.ch/opc/de/classified-compilation/19996136/index.html

GenSuisse. (o. J.). CRISPR/Cas | GEN SUISSE.

Zugriff am 7.5.2019. 

Verfügbar unter: https://www.gensuisse.ch/de/crisprcas

Lahrtz, S. (2018, Juli 25). EuGH bremst neue Gentech-Pflanzen aus | NZZ. 

Zugriff am 7.5.2019. 

Verfügbar unter: https://www.nzz.ch/wissenschaft/eugh-urteil-die-genschere-crispr-faellt-unter-das-europaeische-gentechnikrecht-ld.1406326

Michael Lange. (2018, Juli 25). Hintergründe zum EuGH-Urteil - Was genau macht die Genschere Crispr/Cas? Deutschlandfunk. 

Zugriff am 7.5.2019. 

Verfügbar unter: https://www.deutschlandfunk.de/hintergruende-zum-eugh-urteil-was-genau-macht-die-genschere.676.de.html?dram:article_id=423793

Application of the CRISPR/Cas9 system to modify plants

Overview

In 2012 a new in vitro technique was described which could be one of the biggest discoveries in molecular science in recent years. The new method is called CRISPR/Cas9, where CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 for CRISPR associated. 

Mechanism

CRISPR/Cas9 was discovered in bacteria where it functions as a natural immunity system to fight off viral DNA. When a bacterium detects a foreign DNA strand, it can develop a short copy in form of RNA (called crRNA-spacer sequence) which then gets attached together with another RNA strand (called crRNA repeat sequence) and the protein Cas9. The crRNA-spacer sequence then binds to a certain sequence of bases in the viral DNA and brings the enzyme Cas9 in position to cut the DNA. The enzyme can cut both DNA strands on the opposite sites and leads to blunt ends. The open strands aren’t functionable and the virus is no longer a threat to the bacterium.(Kunin et al.)

In most cells after a DNA was cut, a reparation process begins which often leads to the addition of other bases between the two strands and therefore mutations. (Ochiai)

Figure 1: Mechanism of CASPR/Cas9

Example for its usage

The possible usage of CRISPR/Cas9 is huge. Normally the method is used to knock out certain genes and in plant biotech it can used for example to prevent pre-harvest sprouting or even simpler, to keep a mushroom from turning brown. (Brodwin)

Technical requirements

The method is quite easy to apply in different types of organisms. In plant biotechnology it is possible to insert a viral vector DNA with the coded sequence for the Cas9 protein and the donor molecule into an Agrobacterium which then infects the plant cells and inserts the donor molecule after cutting the DNA with Cas9.(Čermák et al.) Other transfection methods are also already available.

Possible disadvantages in larger number of crops

With CRISPR/Cas9 it's not a necessity to insert new DNA into the cells to change the organism, therefore it was discussed if plants etc. which were treated with CRISPR/Cas9 are per definition Genetic Modified Organisms (GMO) or not. This question is important because in the EU GMO plants are strictly regulated in its usage.

Also, Cas9 can only make blunt DNA ends (other similar systems are able to do sticky ends) which makes it difficult for a possible insert to get implemented in the right direction.

References

Brodwin, Erin. ‘The next Generation of GMO Food Is Here, and It’s Technically Not a GMO’. Business Insider, https://www.businessinsider.com/dupont-crispr-corn-in-stores-in-5-years. Accessed 4 Apr. 2019.

Čermák, Tomáš, et al. ‘High-Frequency, Precise Modification of the Tomato Genome’. Genome Biology, vol. 16, no. 1, Dec. 2015. Crossref, doi:10.1186/s13059-015-0796-9.

Kunin, Victor, et al. ‘Evolutionary Conservation of Sequence and Secondary Structures in CRISPR Repeats’. Genome Biology, vol. 8, no. 4, 2007, p. R61. Crossref, doi:10.1186/gb-2007-8-4-r61.

Ochiai, Hiroshi. ‘Single-Base Pair Genome Editing in Human Cells by Using Site-Specific Endonucleases’. International Journal of Molecular Sciences, vol. 16, no. 9, Sept. 2015, pp. 21128–37. Crossref, doi:10.3390/ijms160921128.

In vitro growth of Bamboo seedlings and possible natural additives

Bamboo raises typically out of rhizomes which can grow horizontally or more in a clumping manner. The conventional propagation method of bamboo is through seeds or by culms which came from offset cutting of the rhizomes. What makes bamboo very special is the fact that all bamboo plants of the same offspring, flower simultaneously at the exact same time and die together afterwards. (Soderstrom and Calderon)
This can lead to the absence of bamboo in large areas followed by negative effects for animals and humans. Therefore, a huge amount of plants has to be reared (up to 100’000 seedlings) in order to have enough for steady cultures in a plantation. (Venkatachalam et al.)

For the proliferation of bamboo culms in a laboratory environment, additives such as the growth hormone BAP (benzyl amino purine), KIN (kinetin), NAA (1-napthalene acetic acid) and IBA (indole-3 butyric acid) were observed to have a positive effect. Several studies tried to find out in which concentrations the plants grew fastest and had the highest rate of survival after they were planted in the field. (Saini et al.)

The results can be used to establish a stable and reliable in vitro propagation procedure.

Coconut water and sucrose were used as growth supplements and both showed very good results in proliferation of the culms.

Anyhow, higher concentrations of coconut water (8 to 10%), showed an inhibition of growth and vitrification. (Venkatachalam et al.) Vitrification is done in some plant species to survive extreme cold temperatures. High concentrations of sugar in the cytoplasm inhibit the formation of ice crystals which could destroy the cell wall.

Several studies were successful in the implementation of a in vitro propagation procedure and showed a possible alternative to the conventional method. To get the whole process of propagation to a larger scale in a laboratory environment is now most likely the trickiest part.

 Pictures are from the internetsite http://www.bamboobotanicals.ca
Link: http://www.bamboobotanicals.ca/html/about-bamboo/bamboo-growth-habits.html

References

Saini, Himanshu, et al. ‘Micropropagation of Himalayan Weeping Bamboo’. American Journal of Plant Sciences, vol. 07, no. 09, 2016, pp. 1317–24. Crossref, doi:10.4236/ajps.2016.79126.

Soderstrom, Thomas R., and Cleofe E. Calderon. ‘A Commentary on the Bamboos (Poaceae: Bambusoideae)’. Biotropica, vol. 11, no. 3, Sept. 1979, p. 161. Crossref, doi:10.2307/2388036.

Venkatachalam, P., et al. ‘Influence of Plant Growth Regulators (PGRs) and Various Additives on in Vitro Plant Propagation of Bambusa Arundinacea (Retz.) Wild: A Recalcitrant Bamboo Species’. Journal of Genetic Engineering and Biotechnology, vol. 13, no. 2, Dec. 2015, pp. 193–200. Crossref, doi:10.1016/j.jgeb.2015.09.006.