Crop improvement is essential to attaining world food security and enhancing nutrition for human beings. Both conventional breeding and modern molecular breeding have contributed to increased crop production and quality. However, the time and resources for breeding practices have been limited. It takes a long time to bring a novel improved crop to the market, and the genetic sources from wild species cannot be always available for crops of our interests. Genome editing technology implemented molecular breeding can overcome those limitations of time and resource by facilitating the specific editing of plant genomes. CRISPR/Cas9 is a rapidly developing technology that has been successfully applied in major crops eg: rice, wheat, maize, barley, Arabidopsis, vegetables, fruits for crop improvement, disease resistance, abiotic stress resistance etc. by gene knockouts, gene replacement, multiplex editing, interrogating gene function, and transcription modulation in plants. As only a short RNA sequence must be synthesized to confer recognition of a new target, CRISPR/Cas9 is a relatively cheap and easy to implement technology that has proven to be extremely versatile. Together with other sequencespecific nucleases, CRISPR/ Cas9 is a game-changing technology that is poised to revolutionize plant breeding and crop engineering.

1. Recent Updates on Application of CRISPR/Cas9
Technique in Agriculture
Seminar Paper Presentation
On
Presented by
Kaniz Fatema
Reg. No: 12-05-2742
MS Student
Department of Biotechnology
Bangabandhu Sheikh Mujibur Rahman Agricultural University
Salna,Gazipur-1706 1

2. Contents
Introduction
 Objectives
 Materials and Methods
 Review of Findings
 Conclusion
2

3. Introduction
 Genome editing
 CRISPR/Cas9 technique
 Advantages of CRISPR/Cas9 over transgenesis and
GMO
 Application of CRISPR/CAS9
3

4. Objectives
The specific objectives of this seminar paper are-
 To review the advances of CRISPR/Cas9
 To review an insight about the recent application
updates of CRISPR/Cas9 technique used in
agriculture.
4

5. Materials and Methods
A Review Paper
Information were collected from
 Various Books, Journals
 Manuscripts, Articles
 Internet browsing &
Compiled and arranged chronologically.
5

6. Review of Findings
6

7. C – Clustered
R- Regularly
I- Interspaced
S- Short
P- Palindromic
R- Repeats
CRISPR/Cas9 Technique
7
 CRISPR stands for
Cas9

8. Figure 1. Mechanism of CRISPR/cas9 is an adaptive immune
system of bacteria
(Source: Baltimore D. et al., 2015)
8

9. Figure 2. A diagram comparing how CRISPR works in the bacterial
immune system and how it works in CRISPR/Cas9 genome editing.
(Source: Sander and Joung, 2014)
9

10. Figure 3. Genome editing with site-specific nucleases. Double-strand
breaks induced by a nuclease at a specific site can be repaired either by
non-homologous end joining (NHEJ) or homologous recombination
(HR).
(Source: Bortesi L., Fischercher, 2015) 10

11. Figure 4. The strategy of using plant genome editing by Cas9/sgRNA
system.
(Source: Hilbeck et al., 2011) 11

12. Character CRISPR/Cas9 Zinc Finger
Nucleases
(ZFNs)
Transcription
factor like effector
nucleases
(TALENs)
Reference
Mode of
action
It works by inducing double-
strand breaks in target DNA or
single-strand DNA nicks (Cas9
nickase).
It can induce
double-strand
breaks in target
DNA
Induces DSBs in
target DNA
Li et al., 2013; Mao
et al., 2013
Off target
effects
These effects can be minimized
by selecting unique crRNA
sequence.
These have
off-target
effects.
Off target effects
cannot be avoided.
Hsu et al., 2013
Multiplexi
ng
Several genes can be edited at
same time. Only Cas9 needed
Highly difficult Very difficult Li et al., 2013; Mao
et al., 2013
Level of
experiment
setup
Easy and very fast procedure
of designing for new target site
Complicated Relatively easy Kumar and Jain,
2014
Table1: Tabular presentation of comparative attributes of plant genome editing
techniques
12

13. The CRISPR/Cas9 Genome-Editing Tool:
Application for improvement in Agriculture
growth improvement,
disease resistance,
abiotic stress tolerance
13

14. Table 2: List of crops genetically edited by CRISPR-Cas9 technique recently
Species Transformation method Target site Desired Character Reference
Rice Agrobacteriam mediated Gn1a Increase grain number
per panicle
Li M. et al. (2016)
Rice Agrobacteriam mediated DEP1 Plant height and
panicle grain size
Li M. et al. (2016)
Rice Agrobacteriam mediated GS3 Seed size Li M. et al. (2016)
Rice Agrobacteriam mediated PA1 Plant height and tiller
number
Li M. et al. (2016)
Rice Particle bombardment ALS Chlorosulfuron
resistance
Sun Y. et al. (2016)
Rice Agrobacterium mediated C-ERF922 Rice blast resistance Wang F. et al. (2016)
Arabidopsis
thaliana
Agrobacterium mediated eIF(iso)4E Potyvirus resistance Pyott D.E. et al.
(2017)
Maize Helium gun MS26, MS45 Male sterile Svitashev S. et al.
(2016)
Maize Particle bombardment ALS2 Herbicide resistance Svitashev S. et al.
(2016)
Maize Particle bombardment AGRS8 Drought tolerance Shi J. et al. (2016)
Maize Plasmid transmitted HVPMI9 Dwarf Lawrenson T. et al.
(2016)
Barley Plasmid transmitted HVPMI9 Dwarf Lawrenson T. et al.
(2016) 14

15. Species Transformation method Target site Desired Character Reference
Wheat Plasmid transmitted MLO Powdery mildew
resistance
Wang Y. et al.(2014)
Tobacco Protoplast transformation NtPDR6 More branching Gao J. et al.(2014)
Soybean Plasmid transmitted FAD2-
1AandFAD2-
1B
Oil quality
improvement
Haun W. et. al.(2014)
Rape seed Agrobacterium mediated ALC Stop seed shattering Braatz J. et al.(2017)
Upland
cotton
Agrobacterium mediated GFP Green fluourescence
protein
Janga M.R. et
al.(2016)
Cirtus Plasmid transmitted CsLOB1 Citrus canker Peng A. et al.(2017)
Grape Protoplast transmitted MLO-7 Powdery mildew Malnoy et al.(2016)
Apple Agrobacterium mediated PDS Clear albino Nishitani C. et
al.(2016)
Tomato RIN Delay ripening Ito Y. et al.(2015)
Populus Agrobacterium mediated PtoPDS albinism Fan D. et al.(2015)
Table 2. List of crops genetically edited by CRISPR-Cas9 technique recently
15

16. CRISPR/cas9 for growth improvement
Species Transformation method Target site Desired
Character
Rice Agrobacteriam mediated Gn1a Increase grain
number per
panicle
Rice Agrobacteriam mediated DEP1 Plant height
and panicle
grain size
Rice Agrobacteriam mediated GS3 Seed size
Rice Agrobacteriam mediated PA1 Plant height
and tiller
number
(Source: Li M.et al., 2016)
Table 3. Details of the four genes mutated in rice plant
16

17. Continued…
Increasing the number of grains in the main panicle
Plant
type
Flower
number
Panicle
length
(cm)
Plant
height
(cm)
WT 104 16 76
Gnla-3 110 16 77
Gn1a-2 184 19 78
Gn1a-10 199 21 82
Table 4. Flower number, panicle length and plant height of Gn1a gene mutated rice
plants
(Source: Li M.et al., 2016)
17

18. Improving long grain phenotype
Continued…
Table 5. Flower number, panicle length and plant height of GS3 gene
mutated rice plants
Plant type Grain
length(mm)
Grain
weight(mg)
WT 6.4 25.7
Gs3-9 6.5 26
gs3-4 8 31
gs3-5 7.5 88
(Source: Li M.et al., 2016)
18

19. Figure 5. pictures showing morphology and grain size of the
GS3 mutant plants
Continued…
(Source: Li M.et al., 2016)
19

20. Table 6. plant height, No. of tiller, panicle length and Flower
number of IPA1 gene mutated rice plants
Continued…
Plant type Plant
height
(cm)
No. of
tiller
Panicle
length
(cm)
No. of
flowers
WT 79 7 17 100
ipa1-3 77 7 18 97
ipa1-5 38 27 8 27
ipa1-12 96 3 21 170
ipa1-21 94 3 22 180
(Source: Li M.et al., 2016)
For lodging resistance and enhanced grain yield
20

21. Figure 6. Morphology of the main panicle of wild type
plants and mutants with the IPA phenotype, grown in
green house
Continued…
(Source: Li M.et al., 2016) 21

22. Dense erect panicle of rice
Pant type Panicle
length
(cm)
Grain size No. of flowers
WT 17.2 25.5 14
dep1-3 18.7 25.1 98
dep1-2 14 24.1 150
Dep1-4 12.4 23.2 168
Continued…
Table 7. panicle length, grain size and Flower number of DEP1gene
mutated rice plants
(Source: Li M.et al., 2016)
22

23. CRISPR-Cas9 targeted mutagenesis in shatter
resistant oilseed rape (Brassica napus L.)
Figure 9. Growth types of CRISPR-Cas9 alc mutants
resemble the wild type while siliques are more shatter
resistant.
Continued…
(Source: Braatz J et. al., 2017)
23

24. Continued…
Figure 10. Results of shatter resistance measurements of alc T2 in
comparison to Haydn. Peak tensile forces are displayed as means of siliques
grouped according to their length.
(Source: Braatz J et. al., 2017)
24

25. Effect of CRISPR/Cas9 in halting the tomato fruit ripening
Continued…
Plant type Phenotype(color) Accumulation
of RIN Protein
Alisa Craig Red +
G1#15A Light orange -
G1#29A Light orange -
G2#31A Red, similar to wild
type
+
G2#13A Moderate orange +
G3#35A Light orange -
G3#42C Delayed but red
eventually
+
G3#54C Light orange -
Table 8. Appearance of mutant fruits harvested five days after the breaker stage
(Source: Ito Y. et al., 2015)
25

26. CRISPR/Cas9 in genome editing of Maize
Continued…
Figure 11. Biallelic mutations in MS45 by RNP result in
male sterile maize.
( Source: Svitashev S. et al., 2016)
26

27. CRISPR/Cas9 in genome editing of soybean
Continued…
Figure 12. The fatty acid profile of FAD2-1 mutant seeds. Fatty acid percentages
were determined for mutant and wild-type seeds:
AABB, wild-type seeds; aaBB, seeds homozygous mutant at FAD2-1A and wild type at
FAD2-1B; AAbb, seeds wild type at FAD2-1A and homozygous mutant at FAD2-1B;
aabbseeds homozygous mutant at both FAD2-1A and FAD2-1B.
(Source:Haun W. et al., 2014)
27

28. CRISPR/cas9 for disease resistance
CRISPR/Cas9 for rice blast resistance by knocking down of
rice ERF gene
Figure 13. Identification of blast resistance in homozygous mutant rice lines and
The blast resistance phenotypes of the mutant rice lines as well as wild-type
plants at the seedling stage. Leaves were detached from the inoculated plants at 7
dpi for photography.
(Source: Wang F. et al., 2016) 28

29. Continued…
Figure 14. Histograms showing the average area of lesions formed on the
third leaves of plants for mutated line and average length of lesions formed
on the inoculated leaves of five tillerings for each line. The values marked
with different letters are significantly different (P < 0.01, Student’s t-test).
(Source: Wang F. et al., 2016) 29

30. CRISPR/cas9 for potyvirus resistance in Arabidopsis by
mutating EIF(iso)4E gene
Continued…
Figure 15. Representative photographs of TuMV-GFP (green
fluorescent protein-expressing Turnip mosaic virus clone)-infected
plants imaged under UV light at 7 days post-infection.
(Source: Pyott D.E. et al., 2017) 30

31. Figure 25. Box plots of dry weights (A)and flowering times
(B) for the CRISPR/Cas9-edited eIF(iso)4E mutants (lines #44,
#65, #68 and #98) alongside a wild-type (WT) plant (#105).
Continued…
(Source: Pyott D.E. et al.,2017) 31

32. CRISPR/cas9 for heritable resistance to powdery mildew in
wheat
Continued…
Figure 16. Micrographs of microcolony formation on the surfaces of
leaves of the indicated genotypes 3 d post inoculation. Powdery
mildew spores and colonies were stained with Coomassie blue. Scale
bars, 200 μm.
32
(Source: Wang Y. et al., 2014)

33. Continued…
Figure 17. Disease symptoms of wild-type (WT) and tamlo-
aabbdd mutant plants. The photograph was taken 7 d after
inoculation in plant. Scale bars, 2 cm.
33
(Source: Wang Y. et al., 2014)

34. CRISPR/Cas9 mediated resistance to Citrus canker
Continued…
Figure 18. Disease lesion area and disease index of leaves of each
mutation line of citrus canker resistance in Wanjincheng orange mutants
investigated at 9 dpi. (Source: Peng A. et al., 2017) 34

35. Figure 19. Growth of Xanthomonas citri subsp. Citri( Xcc) in
leaves of mutant plants
Continued…
(Source: Peng A. et al., 2017) 35

36. CRISPR/cas9 for abiotic resistance
improving maize grain yield under field drought stress conditions
Figure 20. Comparison of the ARGOS8 expression in genome-edited variants
and wild-type maize plants. Relative expression of ARGOS8 in a selection of
maize tissues and stages.. DAP, days after pollination.)
(Source: Shi J. et al., 2016) 36

37. Herbicide (chlorsulfuron )resistance in maize
by introducing ALS1 gene
CRISPR/cas9 for abiotic resistance
(Source: Svitashev S. et. al., 2016)
Figure 21. Maize plants with edited ALS2 allele (left) and the wild type (right)
tested for resistance to chlorsulfuron. A, Four-week-old plants sprayed with
chlorsulfuron (100 mg/L) 3 weeks after spraying. B, embryos germinated on
media with chlorsulfuron (100mg L/L) 14 d after germination.
37

38. Limitations of CEISPR/Cas9 technique
 Cas9 activity
 Target site selection and sgRNA
design
 Delivery methods
 Off-target effects
38

39. Future possibilities of in agriculture
 Genome editing will play very important role in developing new bio-energy
crops, which could give maximum yield on wastelands and changing climate
(Bosch and Hazen, 2013).
 combating abiotic and biotic stress can be the future of crop for Climate resilient
agriculture (Kissoudis et al., 2014; Jain, 2015).
 Direct delivery methods of Cas9 and gRNA using Agrobacterium and Viral
replicons by using nanoparticles can be very useful for simplifying the genome
editing technology (Hiei et al., 2014; Khatodia et al., 2014; Nonaka and Ezura,
2014)
 The generation of large-scale whole-genome targeted sgRNA library for high-
throughput loss-of-function screening applications based on the CRISPRi system
is particularly feasible for model plants in future (Heintze et al., 2013).
39

40. conclusion
• CRISPR/Cas9 technique found become most cheaper, least time
consuming, flexible and powerful platform for genome editing
Compared to Zinc Finger Nucleases (ZFNs) and transcription
activator–like effector nucleases (TALENs).
• The application of CRISPR/Cas9 technique in agriculture found
beneficial to by potentially preventing the spread of diseases, or
supporting agriculture by reversing pesticide and herbicide
resistance in insects and weeds, and control damaging invasive
species.
40

41. 41