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Fostering Collaborations between Indo-Korean

Institutes in Genome Engineering Technology


Background
• India and South Korea - upgrading their bilateral
relationships in science and technology
• Indo-Korea Science and Technology Center (IKST) at
IISc, Bangalore promotes technology and research
partnerships by leveraging complementary strengths and
expertise of the two countries
• This centre functions with a small R&D team in the area of
Computational Material Science
• Possibility of launching joint initiatives in biological
sciences is enormous especially in cutting edge areas viz.,
genomics and genome editing
Genome Editing

Zinc finger nucleases (ZFNs) 1 Module / 3bp

1 Module /1bp
Transcription activator-like
effector nucleases (TALENs)

Clustered regularly interspaced


1 base /1bp
short palindromic repeat/
CRISPR-associated protein
9(CRISPR/Cas9).

(Carroll, 2014)
Comparison of the four types of genome editing tools

(Zhang et al., 2017)


CRISPR/Cas9 system
• Genome editing: Powerful and effective tool in crop
improvement for altering traits that cannot be achieved
through traditional or transgenesis breeding.
• CRISPR/Cas9-induced mutations have much higher
specificity and efficiency
• Foreign sequences can be eliminated during later
generations
• CRISPR/Cas9 system is capable of
• 1) causing mutations at specific loci;
• 2) correcting defective alleles using HDR system and
• 3) integrating transgenes in a pre-determined “safe harbor
loci”
DNA Repair Mechanisms

(Arora and Narula., 2017)


Armed with CRISPR scissors,
Indian scientists look at curing
the incurable
• Indian scientists are using the nanotool to find fixes for
diseases unique to the developing world
• Geneticist Debojyoti Chakraborty (Institute of Genomics
and Integrated Biology, IGIB) has been tinkering with
samples collected from patients of sickle cell anaemia, a
genetically-inherited disease that affects more than 4.4
million people around the world every year
• The blood disorder is widely prevalent among tribal
populations in the Indian states of Chhattisgarh,
Maharashtra, Gujarat, and Odisha.
• CRISPR tool cleaves a target DNA molecule – in this case the
‘haemoglobin B’ locus – where mutation in a single base pair
causes the disease.
• Breaks are fixed by Homology Dependent Repair machinery
based on a new DNA template with the mutation-free gene
sequence.
• Corrected cells were grown in the lab for potential transfusion
back into the patient.
• “Since the cells are the patient’s own, he or she will generally
not reject a graft when the gene-corrected cells are put back into
the body,”
• Proof of concept in human pluripotent stem calls followed by
clinical trials in mouse and then with humans
• Similar attempts are made for correcting defective alleles
causing Beta-thalassemia, Haemophilia etc.,
CRISPR/Cas9 mediated plant genome editing

(Mushtag e al., 2018)


List of genes targeted by genome editing tools for
crop improvement

(Mishra and Zhao., 2018)


List of some crops that are made resistant to diseases via
CRISPR/Cas9 system
Different applications of CRISPR systems

(Ricroch et al., 2017)


Blast resistance in rice

• CRISPR/CAS systems and TALENS frequently have been employed to


achieve disease resistance in rice.

• The pathogen translocates and formulate its virulence proteins, once TAL
effectors internalized into host cell, they bind to promotor elements of host
genes (S) through effector-binding elements (EBEs).

• Result in activation of S gene expression which ultimately developstrong


susceptible reaction between bacterial pathogen and the host plant.

• Small changes at specific sites of TAL-effector binding site in S genes,


ultimately lead to plants which are resistant to bacterial pathogens.

(Shah et al., 2018)


Improved oleic acid level in soybean oil

• TALENs have been utilized to slow down the two fatty acids
desaturase genes activity in soyabean i.e. FAD2 and FAD3
• These are responsible for converting oleic acid
(monosaturated) to linolenic acid (polyunsaturated)
• To create varieties which have high amount of oleic acid
(normal ∼20% vs.∼80%) and less amount of polysaturated
fatty acids including linoleic acid (the usual ∼50% vs. ∼4%)
in their seeds

(Shah et al., 2018)


Herbicide-resistant crops

• Genome editing alter function of ACETOLACTATE


SYNTHASE (ALS) gene by inducing point mutation at
specific locus as this gene is specifically targeted by
imidazolinone (IMI) and sulfonylurea (SU) herbicides.

• Genome editing at target site by induction of point mutation


can be used to generate herbicide tolerant crops.

(Shah et al., 2018)


Declining of phytic acid in maize

• Phosphorus is major content of maize kernel and almost 75%


of it is stored as phytic acid that cannot be easily digested by
human.

• Phytic acid being anti-nutriental compound negatively impact


nutrient uptake and it also impacted environment through its
induction in waste stream.

• Zincfinger nuclease (ZFN) has been used to modify IPK1


gene, which is involved in regulation of biogenesis of phytic
acid. (Shah et al., 2018)
Aroma in rice

• Aromatic rice has primary fragrance compound 2-acetyl-1-


pyrroline (2AP), it is synthesized when non-functional betaine
aldehyde dehydrogenase 2 is present.

• 2AP content in rice grain increased due to disruption of


BADH2 through the use of TALEN methodology

(Shah et al., 2018)


Modulating lycopene content in tomato fruits
through genome editing
Phenotype of tomato fruit at different ripening stages
• Problems associated with CRISPR/Cas9 mediated
mutagenesis
– difficulties in vector construction,
– sgRNA prediction,
– characterizing mutations
– targeting multiple loci in a genome
– Off target mutations
– Regulatory issues
Ethical concerns
• Center around human germline editing.
• human genome editing for reproductive purposes should not
be attempted
• studies that would make gene therapy safe and effective should
continue
• there were about 40 countries that discouraged or banned
research on germ line editing, including 15 nations in Western
Europe, because of ethical and safety
Safety
• off-target effects (edits in the wrong place) and
mosaicism mosaicism (when some cells carry
the edit but others do not)
• risk cannot be justified by the potential benefit
Off-target activity- A major concern
• Improper concentration in the Cas9: sgRNA ratio, the presence of
promiscuous PAM sites and insufficient Cas9 codon optimization
are some of the factors responsible for undesired cleavage of the
DNA regions.

• Strategies like double nicking strategy, SpCas9-FokI strategy and


SpCas9 strategy can be used for minimizing off-target activity.

• Several computational algorithms such as Cas-OFFinder, CRISPR-


P,CRISPR-Plant and CRISPR Primer Designer have been used to
create gene-specific sgRNAs with minimal off-target effects
• Reduction in the expression levels of Cas9/sgRNA reagents
could significantly decrease the off-target effects.
• Next-generation sequencing methods like GUIDE-seq,
Digenome-seq and ChIP-seq can also identify the off-target
sites for Cas9/gRNA .
• Shortening the gRNA spacer sequence to 17–18 nt increases
targeting fidelity.
• Recently, high-fidelity Cas9 variants have been engineered by
substituting 3–4 amino acids. These Cas9 variants have been
quite effective in addressing off-target issue in plants.

(Mishra and Zhao., 2018)


Safety Levels

• Most stringent regulation (in which most of the mutants are subject to the regulations,
whereas only a portion of deletion and insertion mutants fall outside the regulations)
should be initially adopted and
• gradually relaxed because the cultivation and food consumption of genome-edited
crops is likely to increase in the near future.

Araki, M. and Ishii, T./Trends in Plant Science 2015


Proposed Joint Initiatives
• Developing platforms/pipelines/kits/reagents for precise genome
editing in major agricultural crops
• Fine tuning existing methodologies for easy adoption
• Capacity building through training/workshops/exchange visits
Developing platforms/pipelines/kits/reagents for
precise genome editing in major agricultural crops

Indo-Korean Initiative can aim at discovering novel


cas9 or similar endonucleases for precise genome
editing in humans/plants
Cas9 nuclease activity and its modifications

(Arora and Narula., 2017)


Capacity building through
training/workshops/exchange visits

• Student exchange programs


• Faculty exchange programs
• Conduct of joint trainings
Fine tuning existing methodologies

• Developing expertise on various genome


editing tools
• Sharing of resources
• Facilitating development of improved
genetic stocks through genome editing
We can plan for launching an
initiative similar to Indo-US
(GETin)