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Int.J.Curr.Microbiol.App.Sci (2017) 6(11): 5435-5443
5435
Review Article https://doi.org/10.20546/ijcmas.2017.611.520
1
Genome Editing and its Necessity in Agriculture
Asma Majid1, G.A. Parray2, Shabir H. Wani2*, Mojtaba Kordrostami3, N.R.
Sofi2, Showkat A. Waza2, A.B. Shikari2 and Shazia Gulzar1
,Division of Genetics and Plant Breeding, FoA, Wadura, Sopore -193201, SKUAST-K
Jammu and Kashmir, India
2Mountain Research Centre for Field Crops, Khudwani-192102, SKUAST-K,
Jammu and Kashmir, India
3
Faculty of Agricultural Sciences, University of Guilan, Rasht, Iran
Corresponding author*
A B S T R A C T
Introduction
Genome editing is a technique in which DNA
is inserted, deleted or replaced in a genome of
any organism using genome editing tools. It
could be used vastly to edit genome of any
organism. By modifying genome of an
organism we can manipulate the crop growth
features in accordance with our major
purposes as are increasing the production,
eliminating the unfavourable traits and
improving its resistance to various biotic and
abiotic stressors. Although GM crops have
achieved great success in supplementing crop
breeding, but this technique confronts some
technical challenges as it’s expensive and due
to possible unpredictable negative impacts on
environment and human food safety concerns
as well, opposition against them grows
exponentially. Further, plant breeders are
frequently employing mutation breeding
using mutant generators, radiation (gamma
rays or fast neutron) or chemical (ethyl
methane sulfonate or EMS) treatment to
accelerate crop improvement process. This
mutagenic breeding technique expose plants
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 6 Number 11 (2017) pp. 5435-5443
Journal homepage: http://www.ijcmas.com
Genome editing is a technique in which changes are made in the DNA of any organism. A
nuclease promotes breaks in the DNA at a specific sequence which is repaired by several
mechanisms. It is one of the efficient technologies which enable us to change and edit
genome of any organism precisely and accurately. Multiple genome editing technologies
have been employed, including zinc finger nucleases, mega nucleases, clustered regularly
interspaced short palindromic repeats (CRISPR) along with Cas9 protein and transcription
activator-like effector nucleases to modify genome of an organism. These genome editing
technologies exercise several repair systems in which through the applying site-specific
nucleases, areas are altered. SSNs induce double-strand breaks (DSBs) at predetermined
locus in the targeted genome, which can be repaired utilizing well-known procedures such
as non-homologous end-joining or homology-directed repair. Genome editing technique is
reliable for enhancing average yield to fulfil the booming demands of the world’s current
food shortage and to establish a viable and ecologically safe agriculture scheme, to more
precise, productive, economical and eco-friendly.
Ke ywords
Genome editing,
DNA, Protein,
Transcription.
Accepted:
31 September 2017
Available Online:
10 November 2017
Article Info
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to various mutagenic agents that are causing
damage to the plant cells. During the natural
DNA repair process, genetic changes such as
mutations are introduced into the genetic
makeup of plants including genes, these
genetic altercations are random across the
whole genome which result rarely positive
outcomes but more often negative, further
positive outcomes can be strongly linked with
negative outcomes, moreover, natural
mutations recurrence possibility is minute.
Lately, advances in DNA sequencing
technologies, in the respect of cost-effective,
assist conducting elaborate evaluation of
whole living organisms’ genomes which led
to a burst in our understanding of genomics
(Carroll, 2014).Therefore, utilizing genome
editing technology in a highly accurate and
authentic fashion to change and improve the
genome of any organism can be convincingly
accomplished.
Genome editing mechanisms
Genome engineering is interceded by site
specific nucleases that depend on creation of
endonucleases able to engender double
stranded breaks in a targeted genome array.
SSN acquire a DNA-binding domain that
binds to the target sequence (Gaj et al., 2013).
The considered array divided by the site
specific nuclease, that trigger a number of
DNA repair processes at the targeted locus
ranging from deletion to insertion of
transgenes. These mechanisms involve non-
homologous end-joining (NHEJ) in which
two DNA ends ligate together causing erasure
or insertions (InDel) at the break site where
DNA sequences ligates together, thus
resulting in frame shift mutation which
ultimately create a gene knockout. Another
mechanism is homologous recombination
(HR), in which both site specific nucleases
and a DNA repair, arrange array
correspondence to the introduced break site.
DNA ends are ligated to the introduced
pattern which results in gene insertion. The
genome editing replication is depend on large
and influential factors such as stage of cell
cycle, species, tissue type and the applied
frame for editing (Fig. 1).
Genome editing tools
Mega nucleases
Mega nucleases first identified in 80s, they
target large DNA sequences of about 12 to 40
base pairs long which lead them to be highly
specific in the utmost of genomes (Gallagher
et al., 2014). Sequence of this size occurs
generally once in an entire genome which
make them exclusive tools for genome
engineering, since naturally occurring mega
nucleases are uncommon and inadequate to be
used in genome editing, companies were
manipulated mega nucleases in order to be
utilized in genome editing. They are also
called homing endonucleases. Once the DNA
is broken, natural DNA repair processes in the
cells initiated which allowing the insertion of
a justified DNA array. DNA binding domain
and catalytic domain in mega nuclease are
linked so its construction is either expensive
or labour-intensive as compared with other
genome editing tools. Therefore, mega
nucleases have major drawbacks which lead
them to have a considerably low priority as
option amongst genome engineering tools to
work with.
Zinc finger nucleases
Zinc finger nucleases are an artificial genome
editing tool which is constructed by fusing
Zinc finger DNA binding domain with
catalytic domain. Zinc finger nucleases
consists of zinc finger proteins which in the
interest of targeting a particular DNA array to
generate double-stranded breaks by fusing it
with non-specific Fok1 endonuclease they
were manipulated. By taking advantage of
DNA repair mechanism these nucleases can
be used to alter the genome of any organisms
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which is mediated by targeting specific
sequence and inducing break via nonspecific
endonuclease, any zinc finger protein
identifies 3 nucleotides in the target array.
The break induced by zinc finger nuclease is
mediated by site specific nuclease which not
only by non-homologous end joining but
homologous recombination, repair is feasible.
Construction of zinc finger nucleases is
difficult as compared to TALENs and
CRISPR/CAS systems. Further this technique
induces more off target effect in contrast with
other genome editing techniques (Table 1).
Transcription activator-like effector
nucleases
Transcription activator-like effector nucleases
(TALENs) were named as a method of year
by nature methods in 2011 (Baker & Becker
2012). TALENs compose of transcription
activator-like effectors (TALEs) fused with
the non-specific Fok1 endonuclease naturally
found in Flavobacterium okenkoides These
TALEs proteins are naturally exuded by a
bacteria, Xanthomonas spp, which gets bind
to the targeted DNA sequence with the help
of DNA-binding domain. Each duplication
identifies a single nucleotide in target DNA.
TALE protein comprises of N terminal
domain, central repetitive regions and the C
terminal domain. Middle repetitive regions
consists of 34 amino acids which are identical
to each other except for two amino acids at
situations12 and 13 called as Repeat Variable
di-residues (RVD) that determines specificity
of TALEs repeat. Continuous thrust for the
precise, advanced and easier tools resulted in
the development of CRISPR/Cas9.
CRISPR/Cas technology
Clustered regularly interspaced short
palindromic repeat/Cas9 system has been
initially detected in bacteria as a defensive
mechanism versus exterior DNA attack as
bacteriophage. The CRISPR/Cas system
consists of CRISPR RNA (crRNA) and trans-
activating crRNA (tracrRNA) associated with
a Cas9 endonuclease. CRISPR, i.e., Clustered
Regularly Interspaced Short Palindromic
Repeats (CRISPRs) consists of a tandem
direct repeat sequences followed by proto
spacers, i.e. the spaces between these repeat
sequences, both of which are derived from the
invading elements (Kim and Kim, 2014).
Scientists have engineered the two RNA
sequences i.e. crRNA and tracrRNA into one
guide RNA, which is followed by Proto-
spacer Adjacent Motif (PAM) i.e., a 5/-NGG
sequence. The sgRNA has 20 nucleotides at
the 5’end that directs Cas9 to the
complementary target site. The Cas9 protein
is an endonuclease which creates double-
stranded breaks at the target site. This
innovating mechanism of the CRISPR/Cas
system replaced all other mechanisms of
genome editing tools in the year 2012 known
as RNA-guided engineered nucleases.
Cas9 nucleases types
Native Cas9
The double stranded breaks constructed by
native Cas9 which is either by non-
homologous end joining or homologous
directed repair can be repairable.
Cas9 Nickase
It’s developed through mutation in native
Cas9 which induces nicks instead of double
strand breaks. Two Cas9 nickase can be used
simultaneously. This system reduces the off-
target effects.
Inactive dCas9
The nuclease deficient catalytically inactive
mutant version of Cas9 (dCas9) has been
applied for RNA-guided transcription
regulation, as a substitution for genome
engineering (Gilbert et al., 2013; Qi et al.,
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2013). Asan adjusted technique, frequently
and in fact successfully has been employed
for CRISPR interference (CRISPRi) and
CRISPR activator (CRISPRa) as well as for
remarkably effective and precise gene
silencing and activation, accordingly,
adoptingdCas9 with an effector and a sgRNA.
Guide RNAs types
Truncated guide RNAs (truRNA)
They are types of guide RNA with shorter
region of target complementaries which
increases specificity of Cas9 endonuclease.
Ribozyme-gRNA-Ribozyme (RGR)
These are synthetic genes which give rise to
RNA molecule with ribozyme sequence,
owing to their crave to produce considered
guide RNA both in vivo and in vitro, they
might be subjected to self- catalysed break.
Polycistronic tRNA-gRNA (PTG/Cas9)
It’s tandemly arrayed tRNA-gRNA which are
cleaved by tRNA processing system and
targets various locations simultaneously.
CRISPR interference in plants
The CRISPRi has been superb instance of
aRNA-guided, consistent and extremely
effective modulation of the target genes
transcription in plants by fusion of inactivated
dCas9 to effector domains (Larson et al.,
2013). Majorly, this approach utilizes for
transcription adjustment as well as gene
expression, however, recently found
alternative applications in biology, CRISPR
activator (CRISPRa) applied for gene
activation. CRISPRi and CRISPRa libraries
are capable to be applying as versatile tools to
survey the complicated stress-driven
characteristics in plant to conduct functional
genomic analysis. Gilbert et al., (2015)
recognized that the required target site for
effective CRISPRi, comparative to the
transcription start site of a specific gene,
should lie from−50 to +300 base pairs.
Application of genome editing techniques
in crop improvement
Blast resistance in rice
Various genome editing techniques including
TALENS and CRISPR/CAS systems
abundantly have been employed to promote
disease resistance in rice. The main purpose
to develop TALEN technology in rice was its
potential applications inbreeding of disease-
resistant rice varieties. Rice blast disease
occurs due to the interactions within the TAL
effectors from the bacterial parasite
Xanthomonas oryzae pv. oryzae and the
targeted host infection vulnerability (S) genes.
The pathogen formulate and translocates its
virulence proteins, such as TAL effectors into
the host cells, once internalized in the host
cell TAL effectors binds to the promoter
elements [effector-binding elements (EBEs)]
of S genes, activating the expression of S
gene which in turn develops more susceptible
reaction between the host plants and bacterial
pathogen. By making small changes at the
specific TAL-effector binding site in the S
genes, might lead the plants to be resistant to
the bacterial pathogens, because TAL effector
no longer would be capable to identify the
target site of the S gene. Similarly, Targeted
Gene-Editing in Rice has been carried out
through employing CRISPR/Cas9 System.
It’s also substantially cost-effective, precise,
user-friendly, and highly adapted for
numerous gene targeting and high-
performance genome-wide editing with a
comparable or rather superior effectiveness
than ZFNs and TALENs.
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Aroma in rice
The primary fragrance compound in the
aromatic rice is 2-acetyl-1-pyrroline (2AP)
which gets synthesised due to presence of
non-functional betaine aldehyde
dehydrogenase 2. BADH2 inhibits 2AP
synthesis by diverting gama amino-
butyraldehyed which is upstream precursor of
gama amino butyric acid, by disrupting
BADH2 gama-amino butyraldehyde is
converted in acetyl pyrroline, consequently
inducing fragrance in rice.
It has been observed that the disruption of
BADH2 by using TALEN methodology
increased 2AP content in grains from 0 to
0.35–0.75 mg/kg, which was analogous to its
documented content in the positive control
aromatic variety (Shan et al., 2015).
Powdery mildew resistant wheat
Powdery mildew caused by an obligate
biotrophic fangus, Blumeria graminis
f.sp.tritici, which it’s known as the most
severe wheat crop disease, involves in drastic
reduction of yield particularly in temperate
zones. MLO locus being the target site of
pathogen that encodes a G-protein, which
through reverse adjustment the functionality
of plant defence mechanisms, act as facilitator
for the pathogen, orthologous MLO genes are
ubiquitous among all higher plants. In case of
mutant MLO disease inducing property is lost
due to which mildew spreading is impeded
from penetration to the cell wall or at the time
of entry of host. Prof Caixia Gao and her team
at the Chinese Academy of Sciences Institute
of Genetics and Developmental Biology in
Beijing, China, used TALEN (transcription
activator-like effector nuclease) methodology,
to successfully delete function of MLO genes.
It was observed that the omozygous tamlo-
aabbdd plants showed significant resistance to
the powdery mildew infection.
Declining of phytic acid in maize
Maize kernels enriched with phosphorus,
however, the large part of the75%
phosphorous stored as phytic acid which is
not digestible by human. In addition, phytic
acid is an anti-nutriental compound that
negatively confine nutritional intake, farther,
causes harmful impact on environment
through inducing the waste stream. By using
genome engineering procedures, phytic acid
concentration can be reduced. In 2009, Shukla
et al., engineered a ZFN create to modify the
IPK1 gene, one of the genes that is
responsible to regulate the biogenesis of
phytic acid.
Acrylamide-free potatoes
Potatoes after harvesting are stored in cold
chambers to enhance their shelf life, however
during cold storage, starch in potatoes is
degraded, thus when potatoes in frying
process, at high temperatures the cold induced
sugars converts to a brownish colour, at last,
at the end of the process a strong poisonous
acrylamide is formed. Voytas and his
colleagues have designed TALEN constructs
to alter VASCULAR INVERTASE genes,
which by their functions, sucrose content of a
potato can be transformed to glucose and
fructose, they demonstrated that genome-
edited potatoes after cold storage, then in at
high temperatures, generated notably less
brownish pigments and acrylamides as
compared to wild-type potatoes (Clasen et al.,
2015).
Non-browning apples
Genome editing techniques can be
implemented to target polyphenol oxidases
(PPO) genes in apple which are responsible
for inducing brown colour in fresh cut apples,
by mutating PPO genes their browning
inducing activity can be declined.
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Fig.1 Mechanism of genome editing
Deletion or insertion
Table.1 Comparison between various genome editing tools
Mega nucleases
ZFN
TALENS
CRISPR/Cas9
Recognition
site
Between 12 and
40bp
9-18bp
14-20bp
22bp
Efficiency
Low
Medium
Medium
High
Off target
effects
More
More
Limited
No off target
effect
Construction
Highly Difficult
Highly Difficult
Difficult
Easy
Target DNA
recognition
Protein guided.
Protein guided
Protein guided
RNA/DNA
hybrid.
Thus lead to produce apples which remain
fresh for several weeks after they have been
sliced into pieces.
Regulation of ripening in tomato
Ripening in tomato is regulated by RIN genes
which in turn are encoded by MADS-box
transcription factor. To target these regions
within the gene, CRISPR/Cas9 system has
been utilized, it was seen that homozygous
RIN mutant tomato plants remained partially
unripen, in contrast with the wild-type, in fact
it’s verifying the critical role of RIN in the
maturation of tomato (Ito et al., 2015).
Increased oleic acid level in soybean oil
TALENs have been applied to scale down the
activity of the two fatty acids desaturase
genes in soybean, including FAD2 and FAD3,
which converts monosaturated oleic acid to
polyunsaturated linolenic acid to create plants
which their seeds are contain instinctive
amount of monosaturated oleic acid (~80%
vs. the normal ~20%) and low level of
polyunsaturated fatty acid as well as linoleic
acid (~4% vs. the usual ~50%) (Haun et al.,
2014), therefore, produces a healthier and
high quality oil with an improved shelf life.
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Homologous
Recombination
Targeted gene insertion
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Herbicide-resistant crops
Genome editing techniques has provided great
success in generating herbicide tolerant crops.
ZFN mediated genome editing induces a point
mutation at a specific locus in the
ACETOLACTATE SYNTHASE (ALS) gene,
which is mainly targeted by herbicides,
sulfonylurea (SU) and imidazolinone (IMI)
(Townsend et al., 2009). These point mutation
induced at the target site by genome editing
can be useful in generating herbicide-resistant
crops in future as it provides more efficient
and accurate way as replacement of
transgenic breeding.
Coffee without caffeine
Finding caffeine-free coffee has been coffee
breeders goal for years (Borrell 2012), as the
caffeine is highly toxic to humans, further
processes need to be operated to eliminate
caffeine from raw coffee bean which indeed
it’s a challenging and tedious processes, even
sometimes it may generate harmful by-
products, and somehow lead to lessen or take
away other aromatic compounds. In 2003,
Ogita et al., studied RNAi constructs to
silence the responsible gene for biogenesis
caffeine, XANTHOSINE METHYL
TRANSFERASE in Coffea canephora, known
as Rubusta coffee. By practicing genome
editing techniques other caffeine biosynthetic
genes or caffeine transporters can be targeted
in the future, as scientists trying to make
coffee with very little if not without caffeine.
Cotton
The tangibility of targeted gene stacking in
cotton by means of adopting specifically
engineered mega nucleases has been reported
(D’Halluin et al., 2013). In these experiments,
gene present in the embryogenic cells of
cotton possessing a site adjoining to an insect
persistence has been targeted to promote
double stranded breaks via homologous
recombination in the presence of DNA
template possessing two different genes for
herbicide resistance flanked by DNA arrays
with homology to the target site. Roughly2%
of individually modulated callus lines was
indicated to consist no only the precise
insertion but also to pass down the stacked
features to the subsequent off springs.
Herbicidal resistance in tobacco
Mutation in two genes of tobacco i.e. SuRA
and SuRB by adopting zinc finger nucleases
has created herbicide resistant crops
possessing resistance against imidazolinone
and sulfonylurea.
Canola
Zinc finger protein have been used to alter the
oil content in canola seeds by decreasing
palmitic acid and increasing total C18 fatty
acids which is done by enhancing the
multiplication of the two canola b-ketoacyl-
ACP synthase II (KASII) genes in which the
VP16 transcriptional activator domain had
been connected (Gupta et al., 2012). Such
studies have indicated that the engineered
zinc finger protein transcription factors (ZFP-
TFs) can be exerted to adjust genome
organisation in the main food plants and to
obtain advantageous modifications to
ameliorate the agronomically as well as
economically important properties.
Biosafety regulations
Genome editing tools such as ZFNs, TALENs
and CRISPR/Cas9 systems are the nova
techniques employed to accurate mutate in
myriad plant genomes with avoiding of
entering an infinitesimal or no alien DNA.
Woo et al., (2015) determined that naturally
occurring mutations-like can be achieved in
the genome editing by avoiding form
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5442
introducing any exterior DNA with the aim of
preassembled CRISPR/Cas9 proteins.
Kanchiswamy (2016) previously detailed
capabilities and significances of DNA-free
genome editing in crops. Weather genome
edited plants should be considered and were
to put under the resemble regulation to GMOs
or not, propelled scientific communities and
legislators to have an ongoing argument on
this matter. USDA in 2012 presented that
ZFN-edited plants with no transgene
interjection should not be treated with the
established regulations for GMOs. Likewise
in EU, genome edited crops mediated by ZFN
are evaluated under European Community
regulations. Further, the New Zealand
Environmental Protection Authority (EPA)
committee declared that ZFN-1 and TALEN-
mediated engineered plants are not considered
as GMOs (The Mc Guinness Institute 2013).
Recently, Swedish Board of Agriculture
reported that based on EU definition of GMO,
CRISPR-Cas9 cannot be categorized as
genetically modified plants. Lastly, USDA
established no specific regulatory to
cultivation as well as of CRISPR edited
mushroom (Waltz 2016).
Agriculture has achieved great success during
the last decade, however, as result of
population explosion, there are constant
demands while scientists are trying best to
develop new and more efficient technologies
that could help them to have proper responses
to these vital requirements which the present
and the future of mankind depends on..From
production breeding breeders had shifted to
resistance breeding than to resilience breeding
than to genetic engineering approaches and
mutation breeding. As the mutation is a
random process and genetic engineering
approaches are deal with growing number of
regulations and social responsibility, due to
biosafety regulations scientists are these days
mostly concentrated on genome editing as this
technique targets caused less specific
disposition in genome, then low monitoring
and crossing is required, further these new
gene editing technologies are markedly,
accurate, efficient, reliable, economy and
user-friendly. Various genome editing tools
have been utilized which among them
CRISPR/Cas9 technique is the most efficient
one and has abled scientists to create new
opportunities for crop improvement, which
capable of providing ground-breaking
solutions for the food crisis. This is expected
that the improved crops through genome
editing techniques to be generally more
acceptable for consumers as they legally
registered as non-transgenic plants. Applying
of genome editing techniques lawfully,
beyond doubt supply plethora of food to
deteriorate famine in the world, and
interestingly their production will be in a
sustainable manner in the close future.
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How to cite this article:
Asma Majid, G.A. Parray, Shabir H. Wani, Mojtoba Kordostami, N.R. Sofi, Showkat A. Waza,
A.B. Shikari and Shazia Gulzar. 2017. Genome Editing and its Necessity in Agriculture.
Int.J.Curr.Microbiol.App.Sci. 6(11): 5435-5443. doi: https://doi.org/10.20546/ijcmas.2017.611.520
