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)
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Figure 1: Mechanism of CASPR/Cas9
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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.
