Facts

Series

CRISPR-Cas
Genome editing
in plants
Contents
Summary 3

Facts and figures 4

1. CRISPR-Cas - a revolution in genome editing 6

Bacteria protecting themselves against viruses 7

Researchers learn from bacteria 8

Targeted cutting 8

... and natural pasting 10

2. CRISPR-Cas speeds-up research 12

A huge impact 13

Basic research in plants 15

3. Applications of genome editing 16

From plant to crop 17

CRISPR-Cas as precision breeding technique 17

Mutations - source of genetic variation 18

Unprecedented accuracy 21

Extremely versatile 21

4. The difference between genome editing and genetic modification 24

No foreign DNA 25

No selection markers 26

European regulations 26

5. Conclusion 28

References 29
Summary
Genome editing is a revolutionary technology for making rapid and precise changes in the genetic material
of living organisms. This can be done in the DNA of plants, microbes, animals and humans. Using this
technology, scientists can change a specific DNA letter, replace a piece of DNA or switch a selected gene
on or off.

Over the last years, genome editing has transformed life sciences research. This is mainly due to one
very successful form of the technology: CRISPR-Cas. According to the journal Science, CRISPR-Cas was the
scientific breakthrough of the year in 2015.

The CRISPR-Cas system consists of two components: a ‘guide’ and a ‘scissor’. Cas is the molecular scissor:
it is a protein that is guided to a specific place in the genome by a CRISPR RNA molecule (guide RNA
or gRNA). Once at its target, Cas cuts the DNA. This mechanism is not new in itself: bacteria have been
using CRISPR-Cas for a long time to protect themselves against viruses. So rather than being invented by
humans, CRISPR-Cas was devised by nature millions of years ago. People turned this system into a useful
tool to perform well-targeted genome editing.

Just like other genome editing techniques, CRISPR-Cas allows scientists to make very precise changes to
DNA without having to introduce foreign genes in the process. CRISPR-Cas stands out mainly because the
technology is cheaper, faster, more efficient and more versatile than the alternatives. This technique is now
being used in countless labs around the world. Its use has spread beyond basic research because it has
proved to be a very versatile tool for gene therapy and crop improvement. Its first applications in medicine
and agriculture are a fact.

This VIB dossier describes current and emerging applications of CRISPR-Cas technology in agriculture.
This background file is written in an accessible way, so that anyone with a keen interest, regardless of
background, will find it informative. The boxes are for those who want to learn more, but they are not
essential to grasp the general idea.

3
 Facts and figures
DNA is found in the nucleus (‘core’) of each cell. It carries hereditary information and
holds the instructions for what the cell is and can do. The whole of the DNA in the cell
is called the ‘genome’. A single instruction is called a ‘gene’ (see Figure 1 on Page 9).

The genome is a sequence of DNA building blocks or DNA ‘letters’. For example, the
genome of the intestinal bacterium E. coli consists of a sequence of about 3 million
DNA letters. The human genome has 3.2 billion DNA letters.

But humans don’t hold the record in that field. The current genome-size champion is
the canopy plant, Paris japonica, with a genome of 150 billion DNA letters.

Genes are first copied to RNA and then translated into proteins. In addition to a
structural function in the cell, proteins also have a role in chemical conversions,
transport of biomolecules, cellular communication and regulation.

DNA is generally stable. Nevertheless, the sequence of DNA letters can change.
This is a mutation. Mutations can occur naturally in every gene, at any time and in
every cell. Mutations can also be made intentionally by humans - for example by
irradiating the cell or by genome editing.

The word mutation has a negative connotation. Mutations can change the function
of a gene. This can be for the worse: an accumulation of mutations can lead to
cancer in humans and mutations are at the basis of hereditary diseases. However,
the function of a gene can also be improved by a mutation. The fact that we can
digest the protein in cow’s milk, for example, is the result of a mutation. Moreover,
many mutations have no effect whatsoever on the function of a gene. We call these
neutral mutations.

Genome editing in plants

Mutations also create variation within a species. They are therefore essential for life
because without mutations there would be no evolution or biodiversity. Thus, there is
a delicate balance between DNA stability and evolution.

Genome editing is a way of making a specific mutation at a specific, predetermined

location in the genome.

The fact that genome editing has become widespread in recent years is mainly due to
one very successful form of technology: CRISPR-Cas. CRISPR-Cas makes it possible to
modify DNA with unprecedented precision and efficiency.

The magazine Science called CRISPR-Cas the scientific breakthrough of 2015. The
technology was developed from the CRISPR-Cas system that bacteria use to defend
themselves against viruses.

In CRISPR-Cas, CRISPR stands for Clustered Regularly Interspaced Short

Palindromic Repeats and Cas for Crispr associated protein.

5
1 CRISPR-Cas - a revolution
in genome editing
Targeted genome editing is a new development that makes it possible to make changes
in specific genes, whether in bacteria, fungi, plants, animals or humans. This allows the
DNA sequence of a cell or organism to be changed by adding, replacing or removing
DNA letters.
Bacteria protecting themselves
against viruses
The CRISPR story starts in a bacterium. The initial bacteria against bacteriophages. The bacterium
discovery of CRISPR sequences was reported in collects DNA sequences from invading viruses
1987 by Japanese scientists, who investigated the and uses them, in combination with Cas proteins,
genome of the bacterium E. coli. They identified to detect and cut the DNA of these attacking
five identical pieces of DNA that were repeated (see Figure 2).
and were separated by non-repetitive DNA se-
quences of identical size. At that time, these DNA In 2007, using the yogurt-making bacterium Strep-
repetitions were considered a curiosity since they tococcus thermophilus, scientists for the first time
could not be explained. experimentally demonstrated that CRISPR-Cas is
effectively a part of the immune system of bac-
However, when scientists examined the genomes teria6. Repeated exposure of the bacteria to a vi-
of more bacterial species, they kept seeing these rus causes them to develop resistance over time.
same repeated DNA sequences. These species in- This resistance is accompanied by the inclusion of
cluded bacteria used to make cheese and yoghurt viral DNA fragments in the CRISPR region of the
and bacteria that naturally occur in our gut. Since bacteria. When the scientists removed the viral
then, it has been found that more than half of all sections from the CRISPR region, the resistance
bacterial species have CRISPR sequences . 1
disappeared immediately.

The finding that these regular DNA repetitions Various CRISPR-Cas systems have been identified
always occur together with a common group of over the years and, although these systems have
genes, CAS genes, only deepened the mystery. In different characteristics, the mechanism is always
2002 a team of Dutch microbiologists decided to the same: RNA is read from the fragments of DNA
call the region of DNA with these repeats ‘CRISPR’, in the bacterium’s CRISPR library. These pieces of
which is an acronym for ‘clustered regularly inter- CRISPR RNA then go off in search of viral genes
spaced short palindromic repeats’ and called the to bind to. Next, the Cas protein, guided by the
associated genes ‘CAS’ genes, which is short for CRISPR RNA sequence, cuts the viral DNA (see Fi-
CRISPR-associated genes . It quickly became clear
2
gure 2). The collection of fragments of virus DNA
that the proteins encoded by the CAS genes func- therefore serves as a kind of memory. This allows
tion as molecular scissors that can cut DNA. the bacteria to quickly recognize and fight off the
virus the next time it attacks.7,8,9
In 2005, further research showed that the DNA
sequences between the repeats are almost iden-
tical to the genetic material of viruses that infect
bacteria3,4,5. This type of viruses is called bacterio-
phages. The CRISPR region thus appeared to be a
library of viral DNA fragments that the bacterium
has built into its own genome. It was then sugge-
sted that CRISPR-Cas was a system for protecting

7
 Researchers learn from bacteria
Targeted cutting …
The great breakthrough in the use of CRISPR- Later that year (August 2013) five research
Cas as a technology for editing the genomes articles were published that discussed the use
of microbes, plants and animals came in 2012, of CRISPR-Cas in plants16,17,18,19,20. This first set of
when two independent researchers - Jennifer publications about its use in plants showed how
Doudna (UC Berkeley in the US) and Emmanuelle immensely versatile CRISPR-Cas technology was.
Charpentier (then University of Umeå in Sweden, Plant geneticists showed that CRISPR-Cas could be
now at the Max Planck Institute in Germany) - used not only in Arabidopsis thaliana (thale cress) -
showed that you can reprogram the CRISPR-Cas a plant often used by researchers in the laboratory
complex. By modifying the sequence of the CRISPR - but also in food crops such as rice. Later,
RNA molecule, the complex can be made to cut at tomatoes, wheat, maize (corn) and other crops
any desired location in the genome. Care must be were added to the list.
taken to ensure that the sequence of the CRISPR
RNA matches the DNA sequence where the cut is CRISPR-Cas is not the only molecular technology
to be made . 8,9
for editing the genome. Several techniques were
developed that either use molecular scissors
Shortly thereafter, in 2013, five independent other than Cas9 (Cas12, previously called Cpf1,
research teams, including Feng Zang and his Cas13 previously called C2c2, …) or are based on
colleagues from the Broad Institute (MIT, USA), another mechanism, such as oligonucleotide-
showed that the CRISPR-Cas system can also be directed mutagenesis, TALEN technology, and ZFN
used to change the DNA in human cells, mice technology. However, this goes beyond the scope
and zebra fish11,12,13,14,15. The use of CRISPR-Cas in of this dossier. (For an overview, see the VIB Facts
mammalian cells was a pivotal moment in genomic Series issue ‘From plant to crop: the past, present
editing. This was quickly followed by countless and the future of plant breeding’).
publications where the system was used in
different organisms and for different purposes.
2007
First experimental
2013
2002 evidence for CRISPR
immunity
First applications
in mammalian
The terms
‘CRISPR’ and ‘Cas’
2005 2012 cells and plants

1987 are coined

A role as a defense
Programming of
the CRISPR-Cas
First description
mechanism is system becomes
of CRISPR
proposed possible
regions in
bacterial DNA

Genome editing in plants

THE GENOME GOVERNS THE CELL FROM THE NUCLEUS
Cell wall
Nucleus G C
A T
C G
T A

chromosome T A

Cell
DNA

Tomato plant
Figure 1. The genome governs the cell from the nucleus

The nucleus of each cell contains DNA, which is the carrier of hereditary information that holds the instructions for
what a cell is and what it can do. The whole of the DNA in the cell is called the genome.

DNA - a double-stranded molecule in the shape of a helix - is packed into a number of chromosomes. For example,
each cell of a tomato plant has two sets of 12 chromosomes or, to put it another way, two sets of 12 ‘packets of
DNA’. If these chromosomes were to be unrolled and laid end-to-end, they would form a thread about half a meter
long with a diameter of 2 nm (nanometers), or 2 millionths of a millimeter.

As soon as a cell divides, each daughter cell receives the complete genome - all the DNA packets - from the parent
cell. That requires a great deal of DNA copying.

The DNA code consists of 4 ‘letters’: A, T, C and G. The letter A on one DNA strand will always be paired with the
letter T on the other strand, and vice versa. The same is true for the letters C and G. So, when we read one strand,
we also know the letter order of the other - complementary - strand.

The total genome of the rice plant consists of 370 million DNA letters, while that of the potato plant has 840 million
and wheat has 16 billion letters. For comparison, the human genome has 3.2 billion DNA letters. A sequence
of DNA letters encoding an instruction is called a gene.

This instruction can be the recipe for a protein. In other words, the DNA code, or the gene, is read and translated
into a protein via an RNA molecule. Proteins are important in forming the structural parts of the cell, but they
also perform biochemical tasks. They ensure that the cell converts nutrients into energy, produces growth factors,
builds a cell wall, etc. Only a small part of the plant DNA effectively codes for proteins. The rest of the DNA is
important for regulating the transcription of DNA and its translation to proteins, the copying of the DNA, the
maintenance of the structure of the DNA and the chromosomes, and so on.

Occasionally, an error occurs during DNA replication. This is called a mutation. An error in the code of a gene can lead to
a defective protein. A mutation, however, does not need to result in a changed gene product. If mutations, whether they
result in a changed gene product or not, are passed on to the daughter cells, the variation within the species increases.

9
 ... and natural pasting
Breaks in DNA are harmful. Because of this, living In non-homologous end joining, the cell uses
organisms have natural DNA repair mechanisms specific proteins to glue the two ends of the DNA
that detect and repair breaks. This applies just break back together. However, this process is er-
as well to the breaks caused by Cas. When Cas ror-prone and often leads to random mutations at
has cut a DNA strand, one of two natural DNA re- the site of repair, where one or a few DNA letters
pair scenarios can occur: either non-homologous disappear. This can switch off the function of the
DNA end joining or homology-directed DNA re- gene. In many cases, however, that is exactly the
pair. In practice, CRISPR-Cas technology can use intention of the researcher (see the next section).
both mechanisms.

CRISPR-CAS - USED BY BACTERIA TO FEND OFF VIRUSES

(1) Virus invades
bacterial cell

(2) Piece of viral DNA is integrated

into bacterial CRISPR sequence
(4) CRISPR-RNA leads Cas
to a second infection
by the same virus and
disables it by making a cut
(3) Bits of CRISPR sequence
are transcribed into RNA

Figure 2. CRISPR-Cas - used by bacteria to fend off viruses

Viruses consist of a protein coat containing genetic material. A virus multiplies by introducing its genetic material
into a cell (for example a bacterium). Next, the virus uses the hijacked cell’s synthesis mechanisms to produce new
viral proteins and genetic material, which are assembled into new viruses. These can, in turn, infect other cells.

Each time a bacterium is attacked by a virus (1) but survives the attack, the bacterium stores a piece of the virus
DNA in its own genome, specifically in the CRISPR library (2). The bacterium translates this library into CRISPR RNA
molecules (3) that guide the Cas proteins to new incoming viruses that the bacterium recognizes. Cas then cuts up
the viral DNA and, in this way, repels the viral attack (4). (Figure based on reference10)

Genome editing in plants

When the cell uses a new DNA sequence as tem- help of the DNA piece one provides as a template.
plate to repair the break, this is called homo- With this, the DNA around the break is restored to
logy-directed DNA repair or homologous recom- its original state, or a change can be deliberately
bination. For this, however, the ends of this extra built in, depending on the template that is provi-
piece of DNA must largely resemble the DNA se- ded (see Figure 3).
quences around the fracture. The break is then
repaired by replacing the broken region with the

CRISPR-CAS – USED BY PEOPLE TO ALTER DNA

Guide RNA

Matching genomic Cas9

sequence
Genomic DNA

Repair

One or more DNA building blocks

disappear or are added

Figure 3. CRISPR-Cas genome editing

The guide RNA directs the Cas protein to a specific site on the DNA, where it causes a break (1). The cell’s natural repair mechanisms will
try to repair the break. During this natural process errors are often made, resulting in one or more DNA building blocks disappearing or
being added, causing one or more mutations(2).

11
2 CRISPR-Cas speeds-up research
Targeted genome editing is not new. Various techniques for making targeted changes to
DNA exist since several years. What makes CRISPR-Cas so revolutionary is that it is very
cheap, easy to use and can very precisely target specific DNA sequences. Scientists working
at universities and companies therefore massively adopted CRISPR-Cas in their research.
A huge impact
Across the globe, scientists are trying to decipher One important element of this research is reading,
molecular mechanisms and life processes in viru- deciphering and mapping of the complete geno-
ses, bacteria, plants, animals and humans. They mes of organisms. Deciphering genetic codes is
want to know how living things work and how dif- faster than ever thanks to the introduction of new
ferent genes, proteins and biological processes technologies and sequencing instruments. But
interact. This basic research provides insight into reading a genome is not the same as understan-
the origins of diseases such as cancer, brain and ding it. The challenge today is to identify the genes,
nerve diseases, cardiovascular diseases, inflam- determine the function of the corresponding pro-
mations and infections. Over time, this knowledge teins, identify which other non-coding sequences
leads to new medicines, vaccines, diagnostic tests are important in the genome, and so on.
and treatment methods (see the VIB Facts Series
‘Alzheimer’ and ‘Cancer’). With CRISPR-Cas, scientists can quickly identify the
function of a gene or a particular DNA sequence.
Similar insights into growth and disease mecha- CRISPR-Cas allows them to switch off the gene of
nisms of plants are helping scientists to raise the interest and see which characteristics of the cell
yields of agricultural crops, prevent damage by or organism are affected. We call the resulting
diseases and pests, and protect crops against ex- organisms ‘knock-outs’. Multiple genes can also
treme climate conditions such as drought. be studied at the same time by switching them
off simultaneously.

LEGAL WRANGLING
CRISPR-Cas is the subject of a major patent conflict. Various research groups and companies claim that they
made an important contribution to the discovery of CRISPR-Cas and its use as a tool for editing genomes.
This has created a complex patent landscape, with contradictory arguments about ownership, infringement, and
the legality of patents.

Shortly after Jennifer Doudna and Emmanuelle Charpentier showed in 2012 that CRISPR-Cas could be used to
edit DNA9, they each applied for a patent on the technology at the American patent office (on 25 May 2013). The
patent office, however, granted a patent to a competitor, Feng Zhang. He hadn’t submitted his application until
October 2013 but used a faster procedure. Zhang had published the first use of CRISPR-Cas in eukaryotic cells in
201312. Since then, even more researchers have claimed to have been the inventors, including a Lithuanian team
led by Virginijus Siksnys (Vilnius University) and Luciano Marraffini from Rockefeller University (USA). In addition,
hundreds of patents on the use of CRISPR-Cas for specific applications have already been submitted. This has
made the patent situation surrounding CRISPR-Cas very complex.

13
 The stakes involved in the patent dispute are high: whoever gets a patent on CRISPR-Cas can determine whether
(and how much) someone must pay for using the method.

The way in which previous revolutionary breakthroughs in biotechnology - such as recombinant DNA, RNA inter-
ference and PCR - have been dealt with, could lead the way. These technologies can be used freely by academic
and non-commercial research groups, while commercial companies gained access to non-exclusive licenses. This
approach facilitated the broad dissemination of these techniques and could therefore also be seen to offer a
solution in the CRISPR case.

The desired sequence for the CRISPR RNA mo- The supplied CRISPR RNA is inserted into the cells
lecule can be created quickly and cheaply - for together with Cas proteins, after which a muta-
example, by ordering it online from one of the tion is made at a location in the genome homo-
countless DNA and RNA production companies. logous to the CRISPR RNA. Next, the cells carrying
The CRISPR RNA molecules are then delivered a mutation are selected and grown in culture.
to the laboratory by a courier service, just as
you and I order shoes, clothes, books or office
supplies online.

Genome editing in plants

Basic research in plants
The emergence of this new technology to edit The first plants in which DNA changes were suc-
genomes has accelerated plant research. Since cessfully made with CRISPR were Arabidopsis
the introduction of CRISPR-Cas, countless labo- thaliana (thale cress), tobacco, rice and whe-
ratories around the world have used it to iden- at17,18,19,20. This was quickly followed by maize21
tify the functions of plant genes and to change and sorghum22.
plant characteristics. This is mainly done by cau-
sing mutations at a desired location in the plant
DNA, thereby disabling a gene. After the desired
DNA change is made, the researchers can allow
the mutated cells to grow into complete plants.
Plants, after all, have the unique property to grow
a new plant from a single plant cell.

Scientists thus generate a complete genome-edi-

ted plant from one plant cell where the DNA
change took place. For some plant species this
regeneration happens spontaneously, in other
cases the process has to be stimulated by adding
plant hormones that ensure the production of
roots and leaves. In horticulture, techniques such
as grafting and taking cuttings have used this
principle for centuries.

USING CRISPR-CAS TO BETTER UNDERSTAND HUMAN DISEASES

CRISPR-Cas is also widely used in biomedical research to investigate gene functions. Since the completion of the
human genome project, scientists have been looking ever deeper into human cells and tissues. They systematically
try to identify the functions of each of the more than 20,000 genes in our genome. CRISPR-Cas is a great addition
to their toolbox as it allows a gene to be switched off easily in mammalian cells, including human cells in culture.

Scientists often work with disease models to understand diseases. These are experimental animals or cell lines in
which a human disease is mimicked by means of precise genetic changes. Again CRISPR-Cas proved a valuable
tool for this type of research, enabling accurate disease modeling in animals. The laboratory animals commonly
used for this are fruit flies, zebra fish, mice and rats. Disease models are needed to study the onset and progression
of diseases and, at a later stage, to test medicines and other interventions before they are used in people23.

15
3 Applications of genome editing
The plant breeding sector is ready to embrace targeted genome editing for a variety of
reasons. The technology is faster and more precise than traditional plant breeding – also
compared to other genetic modification technology. Moreover, genome editing has the
great advantage that breeders can easily introduce genetic variation into their crops,
which is the starting point for any form of breeding. They can also do this without adding
genes from other organisms - something that has fueled resistance to genetically modified
(GM) crops in several countries. By using genome editing, researchers have already made
disease-resistant wheat and tomatoes, drought-resistant maize, and tomatoes, soya and
canola with a healthier nutrient composition.
From plant to crop
Ever since the emergence of agriculture some Finally, we must reduce the impact of agriculture
10,000 years ago people have been modifying on people and the environment. This can be done
plants (see also the VIB Facts Series issue ‘From by fertilizing in a different way and by using pes-
plant to crop: the past, present and the future ticides more selectively. This benefits the safety
of plant breeding’). They did this by selecting the and health of the farmer and the consumer, and
best performing plants from nature and keeping spares, for example, useful insects.
their seeds for the next sowing. In addition, crops
with interesting features that arose spontaneous- Future plant breeding has a role in all these are-
ly were selected for further breeding. This often as. Natural resistance mechanisms against fungi,
went against natural selection, because the trait bacteria and insects can be incorporated into
was chosen for characteristics that were con- our current high-yield crops. This reduces their
venient for humans, such as a higher yield, larger dependence on plant protection products. Plant
fruits or a more desirable color. The great wealth breeding can also be used to develop crops that
of crops that we grow and eat today is mainly due use water and fertilizer more efficiently.
to this human selection and intervention.

Crop improvement remains essential today. CRISPR-Cas as precision

Worldwide, agriculture faces major challenges:
the climate is becoming more unstable; in cer-
breeding technique
tain agricultural regions, drought or too much Plant breeders have a variety of methods at their
rainfall are gradually making it impossible to cul- disposal: from selective cross-breeding to innova-
tivate the land efficiently. Even small temperature tive genome editing methods.
rises can have a major impact on the yields of
certain crops. During the 20th century new plant breeding tech-
niques based on new scientific insights and tech-
A second challenge is the increasing world popu- nological developments were introduced. For
lation alongside a rapid global expansion of the example, hybrids, in-vitro techniques, and mar-
middle class. This increases the demand for food ker-assisted selection have been a part of bree-
and fodder crops. If we want to meet this challen- ding practices for decades, regardless of the type
ge, production will have to grow in step with the of agriculture the crops are grown in.
increasing demand.
In recent years, additional techniques have been
Breeders try to anticipate these challenges by developed that can play a role in developing new
producing new varieties that have sufficient yields crop varieties with traits that are beneficial for the
while being better adapted to higher tempera- farmer, the environment, the processors, and/
tures, periods of drought and/or growth on soils or the consumer. These techniques are often re-
that are less suitable for agriculture. ferred to collectively as ‘new breeding technolo-
gies’ (see the VIB Facts Series issue ‘From plant

17
 to crop: the past, present and the future of plant gene25. Switching off this gene in other plants also
breeding’). Genome editing is the latest addition gives their flowers a cauliflower-like appearance.
to these breeding techniques.
Mutation-based plant breeding
It is important to note the following: regardless of The greater the genetic variation within a species,
whether these methods have been developed re- the more opportunities there are to find and com-
cently or have existed for thousands of years, all bine desirable characteristics. In addition to sponta-
plant breeding techniques affect the plant’s DNA. neous DNA mutations, plant breeders started to use
mutation breeding in the 1930s to introduce additi-
Mutations - source of onal variation and create new crop traits. This type
genetic variation of breeding uses radiation or chemicals to make
Spontaneous mutations followed changes to plant DNA at a high rate. This increases
by human selection the genetic variation available for plant breeding.
Until the beginning of the 20th century, plant The result of all this irradiation is a large collection of
breeding was mainly an empirical selection pro- seeds with different random DNA mutations. These
cess in which seeds or tubers from the best adapt- seeds are then used in breeding programs to get
ed crops were stored for the following year. This rid of the unwanted mutations and to identify plants
selective cross-breeding was based on sponta- with desirable, improved characteristics.
neous DNA mutations that occur in nature. These
mutations may be due to errors that occur during Traditional mutation breeding has resulted in
the copying of DNA that takes place during cell di- 3,200 improved crop varieties in more than 175
vision or may, for example, arise under the influ- plant species, including rice, maize, wheat, bana-
ence of radiation from the sun. However, not every na, tomato, pumpkin and soya. The striking color
change to the DNA sequence leads to new traits. In of the flesh and the sweet taste of the pink grape-
most cases mutations do not result in changes to fruit is a good example of a new crop characte-
the outward characteristics of the plant. But in cer- ristic created by this form of mutation breeding.
tain situations, changes in a plant’s DNA can result
in new beneficial or detrimental characteristics. Crops obtained via mutation breeding have been
These changes contribute to genetic variation. safely cultivated and eaten for decades. Thanks
to its long history of use and its role in creating
Our ancestors noticed these changes and selec- improved crop varieties, mutation breeding has
ted plants with interesting new characteristics to always and everywhere been seen as a safe and
create crops with maximum benefits for humans. reliable way to produce crops. As a result, pro-
The great diversity that currently exists within the ducts developed through mutation breeding are
cabbage family - known as ‘brassicas’ - is a good exempted from the GMO regulations in Europe.
example of this. All brassicas (cauliflower, Brus-
sels sprouts, kale, broccoli, etc.) are created by The disadvantage of traditional mutation breeding
spontaneous mutations from the same cabba- methods is that they easily produce thousands of
ge-like ancestor. The appearance of a cauliflower, DNA changes, of which only one or a few might
for example, is the result of one change in a single be useful.

Genome editing in plants

CRISPR WAXY MAIZE
The seed company Corteva Agriscience (a merger of the companies Dow, Dupont and Pioneer) has taken the lead
in using CRISPR-Cas technology for crop improvement. In the spring of 2016, the company’s scientists developed
the first commercial crop with this technology: a new generation of waxy maize. While the starch from ordinary
maize kernels consists of 25% amylose and 75% amylopectin, the grains of waxy maize contain almost exclusively
amylopectin (97%). Amylopectin starch is relatively easy to process and is widely used in the food processing in-
dustry and in the production of adhesives. For example, the glue on cardboard boxes and on the adhesive strips
of envelopes are often derived from amylopectin starch. The problem was that the first generation of waxy maize -
developed through traditional breeding - had a lower yield than traditional varieties. This has now been remedied
thanks to CRISPR-Cas24. The researchers at Corteva Agriscience not only succeeded in deleting the waxy gene, they
did this in most of the current elite varieties. This makes it possible to create waxy maize varieties much faster and
in a way that avoids the loss of yield. These maize varieties are expected to appear on the American market in a
few years, pending field trials and regulatory testing.

DROUGHT-RESISTANT CRISPR MAIZE

In addition to a new variety of waxy maize, Corteva Agriscience is using CRISPR-Cas to develop a type of maize
that can withstand periods of drought. To do this, Corteva Agriscience is working together with Jennifer Doudna’s
company, Caribou Biosciences26. CRISPR-Cas was used to change the maize gene ARGOS8 in such a way that it is
transcribed more often, resulting in more ARGOS8 protein in the cells. This protein is involved in the regulation of
the plant stress hormone ethylene. Previous studies have shown that higher production of ARGOS8 protein leads
to a better yield under stressful growth conditions such as drought.

The first field trials with the resulting maize hybrids did indeed show an increase in yield under drought stress
compared to control plants, and no decrease in yield in normal conditions. Additional field trials are currently
being carried out at different locations to assess its commercial potential under various conditions. It is expected
that these drought-resistant maize varieties could come onto the market in 5 to 10 years.

19
 PRECISION BREEDING IN PRACTICE
In general, the CRISPR-Cas breeding process consists of six steps. Let’s take wheat as an example. The wheat
varieties that we grow today are very sensitive to mildew, a fungal disease. With CRISPR-Cas, scientists have now
succeeded in developing a type of wheat that is resistant to mildew.29

The 6 successive steps to achieve this are:

1. Genome study. A successful result of CRISPR-Cas-based breeding is always preceded by a detailed genome
study. The crop characteristic that you want to modify (in this example this is sensitivity to infections with mil-
dew) must first be analyzed in detail at the genetic and molecular level.
2. CRISPR design. Once scientists have determined which DNA changes are required to increase the plant’s fungal
resistance, a CRISPR RNA molecule is designed. This RNA molecule determines the place where the mutation
will be made.
3. Getting CRISPR and Cas into the plant cell. The DNA-cleaving enzymes and the guiding CRISPR RNA
molecules must be introduced into the plant cell either via Agrobacterium transformation, via plant viruses, or
directly as a protein-RNA complex.30,31 In the latter two cases, no genetic material is integrated into plant DNA.
After carrying out their editing task, Cas and CRISPR are spontaneously broken down by the plant cell. The final
result is a plant with the desired mutation(s) in the target gene(s). This resulting plant cannot be distinguished
from a plant that has acquired mutations spontaneously or via traditional mutation breeding.
4. Screening. The next step is to track down the cells or plant tissue pieces in which the CRISPR-Cas system per-
formed the desired change (or changes) correctly. This is often done by means of DNA sequencing techniques
to see whether the change has been successful.
5. Regeneration to a complete plant. The modified cells or plant tissue cultures are then grown to a complete plant.
6. Traditional cross-breeding programs. Finally, the desired mutation is incorporated into elite varieties by
traditional plant breeding methods.

Genome editing in plants

Unprecedented accuracy DNA fragment from a different species. This sec-
Genome editing techniques such as CRISPR-Cas ond application is very similar to crop breeding
are mainly used in plants to induce highly con- by traditional GM technology. There is, however,
trolled, precise DNA mutations. Hence, we speak an important difference: with CRISPR-Cas, the
of ‘precision breeding’. By bringing about an effi- researcher can control precisely where the new
ciently targeted DNA change, a gene in the plant gene is inserted in the plant DNA. With traditional
can be switched off or on. This allows breeders to GM technology scientists have little control over
dampen unwanted characteristics or strengthen where the transgene will be integrated (see Chap-
desirable ones. Such mutations can also occur ter 4 ‘The difference between genome editing and
spontaneously in nature. genetic modification’). In some cases, the location
The advantage of genome editing over traditional of the newly-integrated gene in the genome can
mutation breeding is that only desired mutati- affects the plant’s existing characteristics.
ons are created without additional undesirable
random mutations. Various studies have shown To avoid any unintended side effects due to
that off-target mutations rarely occur during ge- random transgene insertion in GM crops, dif-
nome editing of plants . In case they do occur,
27,28
ferent versions of the modified crop are made
they can be tracked down and crossed out. Ge- instead of just one. These versions differ in the
nome editing can therefore be considered as an genomic location where the extra genes are in-
advanced form of mutation breeding. The result serted into the plant’s genetic material. This was
is the same, we only get there faster and more also the case with the development of, Golden
efficiently. An additional advantage is that CRISPR- Rice - rice that is genetically modified to produce
Cas allows one to change different characteristics provitamin A. The initially chosen version failed to
simultaneously. produce an optimal yield because the inserted
provitamin A genes disrupted the action of an im-
Extremely versatile portant growth gene. This meant that a different
CRISPR-Cas can be used in various ways as version had to be used. This delayed the develop-
a breeding method. ment of Golden Rice by several years (see the VIB
Facts Series ‘Golden Rice’).
Mutation-based plant breeding
CRISPR-Cas is mainly used in plants to make These complications are easily avoided by using
a small change in the existing DNA sequence, genome editing methods such as CRISPR-Cas. In
without integrating foreign DNA. It is therefore a addition, CRISPR-Cas can be used to introduce
modern form of mutation breeding that induces different transgenes at the same location in the
genetic variation in a crop quickly and specifical- genome so that they are passed from the parent
ly. The resulting crop varieties can be identical to to the daughter plant as a single unit.
those produced by traditional breeding methods.

Transgenesis
It is also possible to incorporate a transgene
into a plant with CRISPR-Cas. A transgene is a

21
 MILDEW-RESISTANT WHEAT
The development of wheat that is resistant to the fungal disease mildew is a good example of the power of genome
editing technology. Today, farmers use fungicides to combat mildew. In this mildew-resistant wheat, the genes
responsible for sensitivity to mildew have been disabled so that pesticide use can be greatly reduced29.

Wheat’s sensitivity to mildew is determined by its MLO gene. This gene codes for a protein that the fungus exploits
to invade plant cells. In other words, MLO proteins form a weak point in wheat’s defense against mildew. Disabling
this gene is therefore an attractive way to make the plant resistant. However, the difficulty lies in the size and
complexity of the wheat genome. Bread wheat, for example, has six copies of each gene. To make wheat resistant
to mildew, all six copies of the MLO gene must therefore be switched off. By using radiation or chemicals (traditional
mutation breeding, see earlier) this is simply not feasible as this technique does not target specific genes. Chinese
researchers accepted the challenge to produce mildew resistant wheat by using genome editing and succeeded in
switching off all six MLO genes in the plant.

The American biotechnology company Calyxt plans to develop this wheat commercially. Currently, trials are being
conducted in test fields to see whether the crop trait is robust under open air conditions. At the same time, the
fungal resistance trait is being crossed into various wheat varieties via traditional breeding methods. If all goes
well, these wheat varieties could be sold to farmers by 2022.

Genome editing in plants

MAKING GRAPEFRUIT RESISTANT TO CITRUS CANKER
The cultivation of citrus fruit involves many challenges. One of these is citrus canker, which is caused by the
bacterium Xanthomonas citri. The most effective way to combat the disease is to grow resistant varieties. However,
breeding citrus trees by traditional methods is a challenging and lengthy process.

Researchers at the University of Florida (USA) have succeeded in using CRISPR-Cas to turn off the CsLOB1 gene in
grapefruit plants. The bacterium Xanthomonas citri exploits the encoding protein to colonize the plant. Grapefruit
varieties with a disabled CsLOB1 gene manage to ward off this bacterial infection, which makes them resistant to
citrus canker32.

FROM CRISPR BANANAS TO PEANUTS TO SOYA AND SUGAR BEET ...

AND WHATEVER ELSE IS IN THE PIPELINE
Universities and companies are working on many more crops to obtain useful varieties: wheat with reduced gluten
levels; hypoallergenic peanuts; disease-resistant bananas and sugar beets; mildew-resistant grapes and tomatoes;
soya and canola with a healthier fatty acid composition; and tomatoes with five times more of the antioxidant
lycopene than wild varieties.

23
4 The difference between genome
editing and genetic modification
Just as with GM technology, genome editing is used to purposefully modify one or a few
crop characteristics. However, the similarity stops there.
No foreign DNA
Genetic modification of a plant allows new genetic into the cell as an RNA-protein complex. After
information to be incorporated into its DNA. The the components have done their work and made
new DNA fragment may come from a crossable the desired DNA change, they are simply broken
species or from a species that the crop cannot down in the plant cell. Because CRISPR-Cas does
cross with. The products of this method are called not incorporate any new DNA fragments, it is also
genetically modified crops or GM crops. Over the described as ‘footprint-free’ DNA modification.
years, various GM crops have been developed by
companies and public research institutions. Since Another approach is to introduce the genes
1996, GM varieties of various crops have been encoding the CRISPR-Cas system into the plant via
grown worldwide, both on a large industrial scale Agrobacterium transformation. In this case, foreign
and a small, local scale, especially in North and genes are indeed integrated into the plant DNA.
South America and in Asia . 33
Nevertheless, a final product that does not contain
any additional, foreign genes can be obtained by
Compared with traditional genetic modification, outcrossing the integrated CRISPR-Cas genes in
genome editing is simpler, more efficient and a traditional cross-breeding program. Again, no
targeted. What’s more, it leaves no trace behind. trace of the genome editing is left behind, apart
It is possible to turn genes on or off with CRISPR- from the desired mutation.
Cas without inserting foreign DNA into the plant.
The CRISPR-Cas components can be introduced

Crop variety obtained through

mutation-based breeding
Multiple random mutations in the plant’s DNA Plant genome

Crop variety obtained

through genome editing
Specific mutation in gene of interest

Crop variety obtained through

genetic modification
Foreign DNA introduced into plant genome

Figure 4. Illustration of the differences at DNA level between mutation-based breeding, genome editing, and genetic modification

25
 No selection markers
In traditional genetic modification, a DNA con- genetically modified organisms. Other countries
struct - a sequence of different genes - is intro- - outside Europe - have opted not to put these
duced into the plant’s genome. This construct agricultural crops under such stringent legislation
contains selection marker genes in addition to the (see the ‘Other countries, other choices’ box).
gene of interest. Selection markers make it possi-
ble to easily select plant cells that have integrated Scientists and researchers received the court’s
the gene of interest. ruling in disbelief. They do not understand why
radiation-derived mutants do not fall under these
Frequently used selection markers include genes rules, but the CRISPR-Cas mutants do. CRISPR-
for antibiotic resistance, herbicide tolerance or Cas mutants are, after all, at least as safe, if not
fluorescence. It is a major advance that precision more, as well as much more cost-effective.
breeding with CRISP does not require selection
markers. By reading the DNA sequences of plant Due to this ruling a great deal of much-needed
cells on a large scale, biotechnologists can now precision breeding will be halted - in Europe at
work without the need for markers. least. Twenty years of experience with the legis-
lation on GM crops in Europe has shown that
CRISPR-Cas thus makes it possible to change market authorization for the cultivation of these
a crop characteristic in a highly targeted way, crops is systematically blocked by the EU, even
without the insertion of selection markers, foreign when the European Food Safety Authority (EFSA)
genes, or other genomic scars. has evaluated the crop positively.

The ruling also creates a problem in the enforce-

European regulations ment of the legislation. There are no foolproof
detection methods that can demonstrate the dif-
European regulations, however, do not recognize ference between the genetic changes made us-
this distinction. In the context of plant breeding, ing conventional breeding techniques and those
Europe has two types of regulations. A first one for made by genome editing. It is almost impossible
putting new plant varieties on the market and a se- to check the market access of imported CRISPR-
cond specific regulation for GM crops. Since 2008 Cas crops. However, Europe can still turn the tide
there has been a debate on whether the products by bringing its regulations into line with interna-
of new breeding techniques, such as CRISPR-Cas, tional practice.
fall under the European rules for GM crops.

In July 2018, the European Court of Justice ruled

that agricultural crops in which mutations have
been made with CRISPR-Cas must be regarded as
GM crops.34 They must comply with the extreme-
ly strict conditions of the European directive for

Genome editing in plants

OTHER COUNTRIES, OTHER CHOICES
The US government makes radically different choices when it comes to crops that have been improved by CRIS-
PR-Cas technology. The US Department of Agriculture decided that the new waxy maize (see page 19) does not re-
quire regulatory authorization because the crop does not meet the department’s criteria for a genetically modified
organism. The department has also allowed the Calyxt mildew-resistant wheat (see page 22) to be commercialized
without having to undergo the GM crop regulation process35.

The worldwide success of CRISPR-Cas technology in agriculture will to a large extent depend on the position of
local governments. In addition to the US, Brazil, Argentina, Chile, Japan and Israel also assess the products of
genome editing on a case-by-case basis and CRISPR-Cas-processed crops are not automatically classed as GM
crops. On the contrary, if the crop contains genetic variations that may equally well have been obtained by cross-
ing or through random mutations, they conclude that the crop is non-GMO.

In November 2018, at the request of a group of eight countries, the Committee on Sanitary and Phytosanitary
(SPS) Measures of the World Trade Organization (WTO) issued a memorandum on genome editing. The memo-
randum states that the new instruments for genome editing can significantly reduce the costs and deadlines for
generating new crops, thereby making public researchers and technology companies better able to support local
needs and challenges. This applies in particular to developing countries. The memorandum calls for a globally
harmonized approach to genome editing. An approach based on sound scientific knowledge. The WTO members
that have supported this initiative so far are Argentina, Australia, Brazil, Canada, Colombia, the Dominican Re-
public, Guatemala, Honduras, Jordan, Paraguay, the US, Uruguay and Vietnam36.

27
 5 Conclusion
Genetics has developed at an enormous rate: in less than one human lifetime we have gone from the
discovery of the double helix structure of DNA (1953) by James Watson, Francis Crick, and Rosalind
Franklin to genetic modification with restriction enzymes and PCR in the 1980s, large-scale genome
analysis since 2000, and now the development of genome editing.

Future basic research using CRISPR-Cas will focus on, amongst other things, the development of new
methods for the efficient and safe introduction of Cas proteins and their guiding CRISPR RNAs into
cells and tissues of complex organisms. Today, rapid advances in this technology already allow us to
make unprecedentedly accurate changes in the DNA of almost all living things.

In addition, many new applications are beginning to emerge in healthcare as well as in agriculture.
New crop varieties generated in laboratories are now ready for field trials or are on the verge of
market launch. In healthcare, we have never been closer to a successful implementation of gene
therapy as we are today. All this thanks to the new ‘toolkit’ for genome editing that is CRISPR-Cas.

But the regulatory framework is still far from clear and seems, at least in Europe, to be going the
wrong way. This is a challenge for policy and regulatory authorities. Technologies and their products
evolve rapidly and must be continuously monitored and regulated. The effects they have on the
environment and their risks to human and animal health must be kept to a minimum. Despite this,
regulation should not paralyze innovation and block development of useful products. Government
policy should be proportionate and non-discriminatory.

In addition, a dialog with the end user - in this case the consumer - is important. Two-way
communication also requires scientists and industry to listen to the concerns and arguments of the
consumers. Not only the what, how, and why should be discussed, but above all we have to get
together to discuss and think about which direction we want to take in agriculture.

The price and convenience of the new CRISPR-Cas technology allows for democratic use. Let this
technology become available to as many different plant breeders as possible - breeders who work
with a wide range of crops. Let us help them to enter into dialog with their stakeholders and those
who use their products.

Genome editing in plants

References
1 Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns SJ, Charpentier E,
Haft DH, Horvath P, Moineau S, Mojica FJ, Terns RM, Terns MP, White MF, Yakunin AF, Garrett RA, van der Oost J,
Backofen R, Koonin EV. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol. 2015
Nov;13(11):722-36. doi: 10.1038/nrmicro3569.
2 Jansen R, Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in
prokaryotes. Mol Microbiol. 2002 Mar;43(6):1565-75.
3 Mojica FJ, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic
repeats derive from foreign genetic elements. J Mol Evol. 2005 Feb;60(2):174-82.
4 Pourcel C, Salvignol G, Vergnaud G. CRISPR elements in Yersinia pestis acquire new repeats by preferential
uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.
Microbiology. 2005 Mar;151(Pt 3):653-63.
5 Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome repeats (CRISPRs)
have spacers of extrachromosomal origin. Microbiology. 2005 Aug;151(Pt 8):2551-61.
6 Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides
acquired resistance against viruses in prokaryotes. Science. 2007 Mar 23;315(5819):1709-12.
7 Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J,
Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.
Nature. 2011 Mar 31;471(7340):602-7. doi: 10.1038/nature09886.
8 Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific
DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A. 2012 Sep 25;109(39):E2579-86.
9 Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA
endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21.
doi: 10.1126/science.1225829.
10 Barrangou R, Marraffini LA. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.
Mol Cell. 2014 Apr 24;54(2):234-44. doi: 10.1016/j.molcel.2014.03.011.
11 Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome
engineering via Cas9. Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033.
12 Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome
engineering using CRISPR/Cas systems. Science. 2013 Feb 15;339(6121):819-23. doi: 10.1126/science.1231143.
13 Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided
endonuclease. Nat Biotechnol. 2013 Mar;31(3):230-2. doi: 10.1038/nbt.2507.
14 Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells.
Elife. 2013 Jan 29;2:e00471. doi: 10.7554/eLife.00471.
15 Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Efficient genome
editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013 Mar;31(3):227-9. doi: 10.1038/nbt.2501.
16 Feng Z, Zhang B, Ding W, Liu X, Yang DL, Wei P, Cao F, Zhu S, Zhang F, Mao Y, Zhu JK. Efficient genome
editing in plants using a CRISPR/Cas system. Cell Res. 2013 Oct;23(10):1229-32. doi: 10.1038/cr.2013.114.
17 Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. Multiplex and homologous
recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.
Nat Biotechnol. 2013 Aug;31(8):688-91. doi: 10.1038/nbt.2654.
18 Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. Targeted mutagenesis in the model plant Nicotiana
benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013 Aug;31(8):691-3. doi: 10.1038/nbt.2655.
19 Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C. Targeted genome modification
of crop plants using a CRISPR-Cas system. Nat Biotechnol. 2013 Aug;31(8):686-8. doi: 10.1038/nbt.2650.
20 Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR-Cas system.

29
Mol Plant. 2013 Nov;6(6):1975-83. doi: 10.1093/mp/sst119.
 21 Liang Z, Zhang K, Chen K, Gao C. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. J
Genet Genomics. 2014 Feb 20;41(2):63-8. doi: 10.1016/j.jgg.2013.12.001.
22 Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted
gene modification in Arabidopsis, tobacco, sorghum and rice.
Nucleic Acids Res. 2013 Nov;41(20):e188. doi: 10.1093/nar/gkt780. Epub 2013 Sep 2.
23 VIB Fact Series : Dossier Dierproeven (Animal Testing Dossier).
http://www.vib.be/nl/educatie/Documents/vib_dossier_dierproeven_NL_2018_1003.pdf
24 Chilcoat D, Liu ZB, Sander J. Use of CRISPR/Cas9 for Crop Improvement in Maize and Soybean.
Prog Mol Biol Transl Sci. 2017;149:27-46. doi: 10.1016/bs.pmbts.2017.04.005.
25 Kempin SA, Savidge B, Yanofsky MF. Molecular basis of the cauliflower phenotype in Arabidopsis.
Science. 1995 Jan 27;267(5197):522-5.
26 Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, Hakimi SM, Mo H, Habben JE. ARGOS8 variants generated
by CRISPR-Cas9 improve maize grain yield under field drought stress conditions.
Plant Biotechnol J. 2017 Feb;15(2):207-216. doi: 10.1111/pbi.12603.
27 Tang X, Liu G, Zhou J, Ren Q, You Q, Tian L, Xin X, Zhong Z, Liu B, Zheng X, Zhang D, Malzahn A, Gong Z, Qi Y, Zhang
T, Zhang Y. A large-scale whole-genome sequencing analysis reveals highly specific genome editing by both Cas9
and Cpf1 (Cas12a) nucleases in rice. Genome Biol. 2018 Jul 4;19(1):84. doi: 10.1186/s13059-018-1458-5.
28 Peterson BA, Haak DC, Nishimura MT, Teixeira PJ, James SR, Dangl JL, Nimchuk ZL. Genome-Wide Assessment
of Efficiency and Specificity in CRISPR/Cas9 Mediated Multiple Site Targeting in Arabidopsis.
PLoS One. 2016 Sep 13;11(9):e0162169. doi: 10.1371/journal.pone.0162169.
29 Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL. Simultaneous editing of three homoeoalleles in hexaploid
bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol. 2014 Sep;32(9):947-51.
doi: 10.1038/nbt.2969.
30 Vainstein A, Marton I, Zuker A, Danziger M, Tzfira T. Permanent genome modifications in plant cells
by transient viral vectors. Trends Biotechnol. 2011 Aug;29(8):363-9. doi: 10.1016/j.tibtech.2011.03.007.
31 Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS. DNA-free genome
editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.
Nat Biotechnol. 2015 Nov;33(11):1162-4. doi: 10.1038/nbt.3389.
32 Jia H, Zhang Y, Orbović V, Xu J, White FF, Jones JB, Wang N. Genome editing of the disease
usceptibility gene CsLOB1 in citrus confers resistance to citrus canker.
Plant Biotechnol J. 2017 Jul;15(7):817-823. doi: 10.1111/pbi.12677.
33 ISAAA Brief 53 Global Status of Commercialized Biotech/GM Crops in 2017 -
https://www.isaaa.org/resources/publications/briefs/53/download/isaaa-brief-53-2017.pdf
34 The Court of Justice of the European Union. Organisms obtained by mutagenesis are GMOs and are, in principle,
subject to the obligations laid down by the GMO Directive.
https://curia.europa.eu/jcms/upload/docs/application/pdf/2018-07/cp180111en.pdf
35 Ledford H. Gene-editing surges as US rethinks regulations.
Nature. 2016 Apr 14;532(7598):158-9. doi: 10.1038/532158a.
36 World Trade Organization (WTO) International Statement On Agricultural Applications Of Precision Biotechnology,
26 October 2018, https://docs.wto.org/dol2fe/Pages/FE_Search/FE_S_S009-DP.aspx?language=E&CatalogueId-
List=249838,249823,249748,249641,249507,249371,249321,249324,249267,249265&CurrentCatalogueIdIndex=
6&FullTextHash=&HasEnglishRecord=True&HasFrenchRecord=True&HasSpanishRecord=True

Genome editing in plants

Fundamental research in the life sciences - that is the core activity of VIB.
VIB is an independent research institute where some 1,500 top scientists from
Belgium and abroad carry out pioneering basic research. In doing so, they push
the boundaries of our knowledge of the molecular mechanisms that regulate the
functioning of the human body, plants, and micro-organisms.

Thanks to close collaboration with the Flemish universities UGent (Ghent

University), KU Leuven (University of Leuven), UAntwerp (University of Antwerp),
Vrije Universiteit Brussel and UHasselt (Hasselt University), and a solid investment
program, VIB combines the collective scientific expertise of all its research groups
in a single institute. The results of that research are translated via technology
transfer into concrete applications for society such as new diagnostics, medicines,
treatment methods and agricultural innovations. These applications are
often developed by young start-ups that have arisen from the VIB or through
collaboration with existing companies. In this way, additional employment is
created and we bridge the gap between research and entrepreneurship.

VIB also actively participates in the public debate on biotechnology by developing

and disseminating scientifically substantiated information.

For more information, go to www.vib.be

VIB
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R.E. Jo Bury, Rijvisschestraat 120, 9052 Ghent, Belgium - D/2019/12.267/1