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.