Genome editing has emerged as a novel strategy for crop improvement using site-specific endonucleases to introduce precise double stranded breaks in plant genomes. This triggers DNA repair mechanisms of nonhomologous end-joining or homology-directed repair that can result in targeted mutagenesis or genome editing. Three classes of endonucleases have been used - zinc finger nucleases, TALENs, and CRISPR/Cas9. CRISPR/Cas9 involves a Cas9 endonuclease guided by a short RNA to cleave target DNA sequences. Case studies demonstrate using CRISPR/Cas9 to edit multiple loci in tomato to improve traits. Genome editing holds potential for agricultural crop improvement, disease management and functional gen

1. IndianAgriculturalResearchInstitute
Genome editing for vegetable crop
improvement
Suman Lata
Scientist (Ag. Biotechnology)
Division of Vegetable Science
ICAR-IARI, New Delhi-12

2. Emerged as a novel strategy for crop improvement
 This technique involves site specific endonucleases (SSNs) to
introduce double stranded breaks (DSB) at precise position in
the genome to initiate DNA repair mechanism.
Two kind of repair system:
Nonhomologous end-joining (NHEJ)
Homology-directed repair (HDR)
Repair mechanism Joins the broken DNA strands which leads
to editing of genome
Targeted mutagenesis or Genome editing
IndianAgriculturalResearchInstitute

3. Three major classes of endonucleases have been used:
Zinc-finger nucleases (ZFNs)
Transcription activator-like effector nucleases
(TALENs)
Clustered regularly interspaced short palindromic
repeats (CRISPR)/Cas(CRISPR associated)
IndianAgriculturalResearchInstitute
Types of Endonucleases

4. IndianAgriculturalResearchInstitute
Zinc-finger nucleases (ZFNs)
ZFNs are made of:
I. A zinc-finger protein (ZFP) domain which is sequence-
specific DNA-binding domain
II. A nonspecific DNA cleavage domain from the type II
restriction enzyme FokI.
The FokI nuclease act in dimeric form to cleave DNA
Two different ZFN monomers, each binding to a different
strand, are required for an active nuclease.
ZFN monomers which flank the target site and are separated
by a 5- to 7-bp spacer sequence.
(Lee et al, 2016)

5. IndianAgriculturalResearchInstitute
Zinc-finger nucleases (ZFNs) structure
Fig. Schematic representation of a ZFN dimer bound to DNA. Each ZFN is composed
of a zinc-finger protein at the amino terminus and the FokI nuclease domain at the
carboxyl terminus.
(Lee et al, 2016)

6. IndianAgriculturalResearchInstitute
Transcription activator-like effector nucleases
(TALENS)
TALENs are artificial nucleases synthesized by sequence-specific
DNA-binding domain and a nonspecific DNA cleavage domain derived
from FokI
DNA-binding domain known as transcription activator-like effectors
(TALEs)
TALEs are consist of tandem repeats of 33–35 amino acid
repeats, each of which binds to single base pair target DNA
Target sequences of TALEN pairs are 30–40 bp in length, with 12- to
21-bp spacer.
TALENs can be used to target almost any given DNA sequence,
which is a critical advantage over other types of nucleases

7. IndianAgriculturalResearchInstitute Structure of transcription activator-like effector
nucleases (TALENS)
(Abdallah et al. 2015)
Fig. schematic diagram of a TALEN pair. Each TALEN has
transcription activator-like effectors (TALEs) at the amino
terminus and the FokI nuclease domain at the carboxyl
terminus.

8. IndianAgriculturalResearchInstitute Clustered regularly interspaced short palindromic
repeats (CRISPR)/Cas(CRISPR associated)
(Lee et al, 2016)
CRISPR-Cas9 as an adaptive immune system in bacteria and
Archaea (Makarova et al. 2006)
 The Cas9 endonuclease from S. pyogenes can cleave a 23-bp
target DNA sequence of
20-bp guide sequence
a 5’-NGG-3’ sequence known as protospacer adjacent
motif (PAM)
Cas9 proteins derived from species other than S. pyogenes
recognize different PAM sequences

9. Fig. :The CRISPR/Cas9 system for gene editing (Weeks et al, 2016)
IndianAgriculturalResearchInstitute Mechanism

10. IndianAgriculturalResearchInstitute
ZFN TALEN RGEN
(CRISPR/Cas9)
Recognition site 18–36 bp per ZFN
pair
30–40 bp per
TALEN pair
22 bp (20-bp guide sequence + 2-
bp protospacer adjacent motif
(PAM) from Streptococcus
pyogenes)
Restriction in target
site
G-rich Start with T End with an NGG
Sequence Success
rate
Low High High
Off-target effects High Low Variable
Cytotoxicity Variable to high Low Low
Size ~1 kb*2 ~3 kb*2 4.2 kb (Cas9 from Streptococcus
pyogenes) + 0.1 kb (sgRNA)
Ease of engineering Difficult Moderate Easy
Ease of multiplexing Low Low High
Table: Comparison of three classes of designed nucleases
(Lee et al, 2016)

11. IndianAgriculturalResearchInstitute
Advantages of CRISPR/Cas over ZFNs and TALENs
Cost-effective and easy-to-use technology
Precisely and efficiently target or edit genes across
organisms
Simple and feasible preparation compared to ZFN and TALEN
Availability of wide range of vectors targeting many genes
Multiplex genome editing is relatively easy using Cas9
nucleases

12. IndianAgriculturalResearchInstitute
Steps for CRISPR/Cas9 genome editing
Agrobacterium mediated transformation of target gene -Cas9
constructs in desired crop to generate Cas9 transgenics
Generation of
CRISPR/Cas9
constructsCloning of sgRNAs in a shuttle vector
Subcloning of target -gRNAs-scaffold RNA
cassette in Cas9 binary vector
In silico analysis of target gene to design sgRNAs

13. Cas9 T0 transgenics
Generation of Cas9 T1
transgenics
Development of Cas9 T2
transgenicsStability studies of mutations induced by
Cas9
Molecular analysis:
•Confirmation of full gene - CRISPR/Cas9
construct by PCR
•Mutation detection by sequencing,
restriction site loss analysis
•T7 endonuclease I (T7EI) assay
•Estimation of target gene transcript by Real
time PCR

14. IndianAgriculturalResearchInstitute Case study 1
•In this study CRISPR–Cas9 strategy to combine agronomically desirable traits
with useful traits present in wild lines has been attempted.

15. IndianAgriculturalResearchInstitute
Cont.
•Editing of six loci have been reported i.e. general plant growth habit
(SELFPRUNING), fruit shape (OVATE) and size (FASCIATED and FRUIT
WEIGHT 2.2) fruit number (MULTIFLORA), and nutritional quality
(LYCOPENE BETA CYCLASE).
•Engineered S. pimpinellifolium morphology was altered, together with the
size, number and nutritional value of the fruits.
•Compared with the wild parent, the engineered lines have a threefold
increase in fruit size and a tenfold increase in fruit number.
•Fruit lycopene accumulation is improved by 500% compared with the
widely cultivated S. lycopersicum.
Zsögön et al,2018

16. IndianAgriculturalResearchInstitute Case study 2
•In this study CRISPR/Cas9 system strategy has been attempted
to generate parthenocarpic tomato.

17. IndianAgriculturalResearchInstitute
Cont.
•SlIAA9—a key gene controlling parthenocarpy was targeted using
CRISPR/Cas9 system
•Mutation rates of up to 100% obtained in the T0 generation.
•Regenerated mutants exhibited morphological changes in leaf shape and
seedless fruit—a characteristic of parthenocarpic tomato
• The segregated next generation (T1) also showed a severe phenotype
associated with the homozygous mutated genome.
•The CRISPR/Cas9 system developed to produce parthenocarpic tomato can
be applied in a wide variety of cultivars, major horticultural crops.
Ueta et al,2017

18. IndianAgriculturalResearchInstitute Case study 3
•In this study CRISPR/Cas9 system was used to knock-out
Solanum tuberosum transcription factor gene StMYB44.

19. IndianAgriculturalResearchInstitute
Examples of Genes edited in horticultural crops
Crop Gene edited Reference
Solanum lycopersicum ARGONAUTE7 (SlAGO7)
Ripening inhibitor (RIN)
SlAGAMOUS-LIKE 6 (SlAGL6)
Brooks et al., 2014
Ali et al., 2015
Klap et al., 2017
Solanum tuberosum StMYB44 Zhou et al., 2017
Watermelon Phytoene desaturase (ClPDS) Tian et al., 2017
Cucumis sativus Eukaryotic translation initiation
factor 4E (eIF4E)
Chandrasekaran et al.,
2016
Lactuca sativa BRASSINOSTEROID INSENSITIVE 2
(BIN2)
Woo et al., 2015
Brassica oleracea Gibberellin3-beta-dioxygenase 1 Lawrenson et al., 2015

20. IndianAgriculturalResearchInstitute
Perspective
Genome editing is an easy, cost effective and
efficient technique
The applications of this technique involves
•Agricultural crop improvement
•Disease management
•drug development
•Functional genomics

21. IndianAgriculturalResearchInstitute
Thank
you