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March 2018
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Seed
Special
How does cutting DNA
help breeding?
But how can just cutting DNA, result in
a targeted trait introduction (mutation)?
This is where the plant’s (actually any
organism) natural DNA repair mecha-
nisms come into play. During day-to-day
activities or through environmental influ-
ences, DNA strands may naturally break.
Therefore, each cell has the capacity to fix
such breaks using one of two processes:
Non-homologous end joining (NHEJ) or ho-
mologous repair (HR) (
Figure 2
).
The NHEJ system can fix the break by
just joining the two DNA strands again, or
through an error-prone system that naturally
results in small DNA mutations (changes) by
either adding or removing small DNA piec-
es. The HR system uses an existing copy of
the DNA as a template to precisely correct
an error.
Scientists found that when a double strand-
ed break is introduced into a DNA strand,
one of these two mechanisms would be
activated naturally to repair the break. Most
often the NHEJ repair system would be ac-
tivated, to either join the DNA strands again
– thus reforming the original DNA sequence
– or introducing a mutation by deleting or
inserting DNA pieces.
A non-mutational repair results in rebuilding
of the original targeted site, thus allowing
the CRISPR/Cas system or other-directed
nucleases to cut it again. A mutation (DNA
insert or deletion), changes this target site,
making it non-recognisable to the nucle-
ases. HR is used for more precise changes
(mutations) when a ‘correct DNA template’
is provided with the site directed nucleases
from which the cell can ‘copy’ from.
This provides the breeder with the tools to
‘knock-out’ (switch a gene/trait off) a gene
or to even change a single DNA base, there-
by allowing the introduction of a new trait
or edited target gene. This is very similar, if
not identical, to traditional mutation breed-
ing in the past, but it is much more targeted
for a specific trait of interest. ‘Knocking’ a
trait out is currently considered easier than
introducing one. This tool is seen as vital
technology going forward which will have
the largest impact during early generations
of the crop development process.
What it means for the
producer or end user
This technology will be used with increas-
ing frequency in the breeding process in the
future to generate new improved higher-
yielding crop cultivars in half the time. It
will depend on which private companies/
research institutions are willing to invest in
it for the long-term and how it will be regu-
lated.
This tool has already been used success-
fully internationally in the development
of improved cultivars in crops like maize,
wheat, rice, tomato, soybean, with target
traits relating to nutritional quality, drought
tolerance, disease and herbicide resistance.
It has been successfully used on a number
of important crops to introduce herbicide re-
sistance to chlorsulfuron by means of single
DNA base change. Indeed, such herbicide
resistance has already been introduced into
maize, tomato and rice using the CRISPR
technology.
The technology is currently being used in
livestock research to promote animal health
and well-being, with CRISPR/Cas aided
dehorning already shown to work in dairy
cattle.
Safety first
With all these new plant breeding tools
and activities happening around the world,
one would expect to see large numbers of
improved crops being released already.
However, this is not the case. Why?
The success and speed at which the new
tools were developed and released caught
the regulatory bodies across the world off
guard. Many countries are just not sure
how the technologies should be regulated
or even if they should for certain usages.
What is especially concerning is that literally
anyone can experiment with the CRISPR/
Cas system.
This is a real possibility as many CRISPR/
Cas tutorial videos and readymade sys-
tem kits are readily available for purchase
online. Someone can experiment with the
CRISPR/Cas system in their backyard or
‘garage lab’ for good or bad. This is further
complicated since some of the technologies
could also be used for transgene events, i.e.
generating genetically modified organisms
(GMOs) – which are indeed regulated in
many countries, including South Africa.
Food for thought
However, how do you regulate a mutation
that was induced using the existing natural
mechanisms (HR or NHEJ) within the organ-
ism – either directed using the new breed-
ing tools, or induced using non-regulated,
random mutagenesis technologies or even
occurring naturally – especially if you can-
not determine the route taken by looking
at the end product only. The USA is taking
the lead, with a CRISPR/Cas9 edited, non-
browning longer shelf life mushroom re-
cently approved for limited release.
Targeted breeding technologies
Figure 2: Natural DNA repair mechanisms used by directed nucleases (e.g. CRISPR/Cas9) to introduce
mutations at a targeted DNA site.