Genome editing in crop improvement one of the desirable biotechnology concept. It is useful for the production of new varieties against resistance to diseases and insect pests

1. Siddu Lakshmi Prasanna
Ph.D. Scholar
Department of Plant Pathology
CRISPR/Cas 9 - Role In Enhancing Plant Disease Resistance
1

2. Contents
❑ Background
❑ Introduction
• Timeline of CRISPR
• What is CRISPR
• Components
• Types and classes of CRISPPR
• Delivery of CRISPR/Cas Reagents To Plants
• Applications of CRISPR
• Flow of CRISPR/Cas9 mediated plant genome editing
❑ Case studies
❑ Advantages and limitations
❑ Conclusion
2
CRISPR/Cas 9??????

3. Comparison of breeding methods used in modern agriculture.
(Chen et al., 2019)
3

4. Types of genome editing tools
4
(Arora and Narula., 2017)

5. 5
Difference between ZFN’s, TALEN’s and CRISPR

6. (Nidhi et al., 2021)
CRISPR/Cas Timeline and its development
6

7. Jennifer Doudna Emmanuelle Charpentier
7

8. • CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Sequences) system as bacterial immune system
and magnificent tool for plant editing which are a family of DNA sequences found in the genomes of bacteria and
archaea.
• The CRISPR-Cas9 system is a RNA-mediated sequence-specific adaptive immune system of the prokaryotes, which
provide protection against bacteriophages.
• C- Clustered
• R- regularly
• I- interspaced
• S- short
• P- palindromic
• R- repeats
8
Introduction
Figure1: CRISPR Locus structure

9. • CRISPRs were first discovered downstream of the alkaline phosphatase isozyme gene (iap) in Escherichia coli (Ishino et al.,
1987).
• Palindromic repeats are separated by short (32 to 36 bp) sequences derived from the DNA of viruses that have previously
infected the cell or its predecessors.
• These virus-derived sequences integrated into the bacterial genome provide a memory system of previous virus infection.
• Once integrated into the genome, CRISPRs are transcribed and the virus-derived sequences form short guide RNAs that
are bound by CRISPR associated protein 9 (Cas9) which is a DNA endonuclease.
• In bacteria and archaea, the natural role of the CRISPR-Cas9 system is to provide adaptive antiviral immunity against DNA
viruses.
• Binary complexes formed by guide RNA-Cas9 recognize and cleave DNA of incoming viruses with sequence similarity to the
guide RNA
9

10. Components of CRISPR/Cas9
• The important components in the system
include Cas9, gRNA and PAM.
• The nuclease Cas9 acts as a molecular
scissors to cut the DNA strands.
• The gRNA directs the Cas9 to cleave the
DNA at a specific position.
• Protospacer Adjacent Motif (PAM) –is
required for a Cas nuclease to cut and is
generally found 3-4 nucleotides
downstream from the cut site and
localized on the non-target DNA
strand, directly downstream of the
target DNA sequence
(Khan et al., 2018)
10

11. 11
Figure 2: Adaptive immune system of Streptococcus pyogenes against bacteriophages

12. 12
Figure 3: Difference between Natural CRISPR and Engineered CRISPR system

13. 13
(Hullahalli et al., 2015)
Figure 4: CRISPR Locus architecture in E. faecalis

14. (Savitskaya et al., 2016)
Types and classes in CRISPR technology
14
• On the basis of the Cas genes and the nature of the interference complex, CRISPR/Cas systems have been divided
into two classes that have been further subdivided into six types based on their signature Cas genes.
• Class 1 CRISPR/Cas systems (types I, III, and IV) employ multi-Cas protein complexes for interference, whereas class
2 systems (types II, V, and VI) accomplish interference with single effector proteins in complex with CRISPR RNAs
(crRNAs).

15. 15
CRISPR/Cas systems for genome editing and other manipulations
(Chen et al., 2019)

16. Delivery of CRISPR/Cas Reagents To Plants
• CRISPR-mediated editing reagents, including DNA, RNA, and ribonucleoproteins (RNPs), can be delivered into
plant cells by protoplast transfection, Agrobacterium-mediated transfer DNA (T-DNA) transformation, or
particle bombardment.
• Protoplast transfection is normally used for transient expression, whereas Agrobacterium-mediated transformation
and particle bombardment are the two major delivery methods for the production of edited plants.
16
(Chen et al., 2019)

17. (Arora and Narula., 2017)
Applications of CRISPR/Cas9
17

18. 18
The basic flow chart of CRISPR/Cas9 editing of target genes.
(Liu et al., 2017)

19. 19

20. Objective: To improve resistance against rice blast via CRISPR/Cas9-targeted knockout of the ethylene
responsive factors (ERF) transcription factor gene OsERF922 in Kuiku131, a japonica rice variety widely
cultivated in northern China
(Wang et al., 2016)
20

21. Materials and methods:
• Japonica rice variety Kuiku131
• C-ERF922-expressing vector (pC-ERF922)
• Cas9 gene-specific PCR primers Cas9p-F and Cas9p-R
• The target site sequence-containing chimeric primers were cloned into the sgRNA
expression cassette pYLsgRNA-U6a at a BsaI site
• The Cas9/sgRNA-expressing binary vectors (pC-ERF922, pC-ERF922S1S2 and pC-
ERF922S1S2S3) were transformed into an Agrobacterium tumefaciens strain EHA105 by
electroporation
21
(Wang et al., 2016)

22. 22
(Wang et al., 2016)
(A)Schematic diagram of OsERF922 gene structure and the C-
ERF922 target site (ERF922-S2).
(B) Schematic diagram of the pC-ERF922 construct for expressing
the CRISPR/Cas9 protein C-ERF922.
(C) Nucleotide sequences at the target site in the 7 T0 mutant rice
plants.
Figure 1: CRISPR/Cas9-induced OsERF922 gene modification in rice

23. 23
(Wang et al., 2016)
Table 1: Ratios of mutant genotype and mutation types at the target site (ERF922-S2) in T0 mutant plants

24. Figure 2: PCR based identification of T-DNA-free rice mutant plants
24
(Wang et al., 2016)
T-DNA-free plants carrying the desired
gene modifications can be acquired through
genetic segregation

25. 25
(Wang et al., 2016)
Table 2: Segregation and types of C-ERF922 induced mutations in the target and
their transmission to subsequent generations

26. Figure 3: Identification of blast resistance in homozygous
mutant rice lines
26
(Wang et al., 2016)
(A) Nucleotide sequences of the
target site in the 6 homozygous T2
mutant rice lines used for pathogen
inoculation.
(B) The blast resistance phenotypes
at the seedling stage.
(C) average area of lesions formed
on the third leaves of 10 plants for
each line.
(D) Blast resistance phenotypes at
the tillering stage.
(E) average length of lesions formed
on the inoculated leaves of five
tillerings for each line.

27. 27
(Wang et al., 2016)
Table 3: Analysis of the agronomic traits of 6 homozygous T2 mutant rice lines

28. Conclusion:
28
• Inoculation with M. oryzae revealed that blast resistance in the T2 homozygous mutant lines tested was significantly
enhanced compared with that of wildtype plants at both the seedling and tillering stages
• There was no significant difference between T2 homozygous mutant lines and wild-type plants with respect to the
agronomic traits, such as plant height, flag leaf length and width, the number of productive panicles, panicle length, the
number of grains per panicle, seed setting rate, and thousand seed weight.
• This study provides a successful example of improving rice blast resistance using CRISPR/Cas9 technology.
(Wang et al., 2016)

29. Plant species Fungus Target
gene
Gene function Strategy Reference
Triticum
aestivum
Powdery mildew
(Blumeria graminis f.
sp. tritici)
MLO-A1 Susceptibility (S) gene
involved in powdery
mildew disease
Particle bombardment of immature
wheat embryos with Cas9/gRNA
expression plasmid vectors
Wang et al., 2014
Solanum
lycopersicum
Powdery mildew
(Oidium
neolycopersici)
MLO1 Major responsible for
powdery mildew
vulnerability
Agrobacterium-mediated transformation
of cotyledons with Cas9/gRNA
expression plasmid vectors
Nekrasov et al.,
2017
Vitis vinifera Powdery mildew
(Erysiphe necator)
MLO-7 Susceptibility (S) gene
involved in powdery
mildew disease
PEG-mediated protoplast transformation
with CRISPR ribonucleoproteins
Malnoy et al.,
2016
Vitis vinifera Grey mold (Botrytis
cinerea)
WRKY5
2
Transcription factor
involved in response to
biotic stress
Agrobacterium-mediated transformation
of proembryonal masses with
Cas9/gRNA expression binary vectors
Wang et al., 2018
Theobroma
cacao
Black pod disease
(Phytophthora
tropicalis)
NPR3 Regulator of the immune
system
Agrobacterium-mediated transient
transformation of stage C leaves with
Cas9/gRNA expression binary vectors
Fister et al., 2018
Oryza sativa
L. japonica
Rice blast disease
(Magnaporthe
oryzae)
SEC3A Subunit of the exocyst
complex
Protoplast transformation with
Cas9/gRNA expression binary vectors
Ma et al., 2018
Oryza sativa
L. japonica
Rice blast disease
(Magnaporthe
oryzae)
ERF922 Transcription factor
implicated in multiple
stress responses
Agrobacterium-mediated transformation
of embryogenic calli with Cas9/gRNA
expression binary vectors
Wang et al., 2016
CRISPR/Cas9 applications for fungal resistance.
29

30. Jeyabharathy Chandrasekaran, Marina Brumin, Dalia Wolf, Diana Leibman, Chen
Klap, Mali Pearlsman, Amir Sherman, Tzahi Arazi and Amit Gal‐On
Development of broad virus resistance in non‐transgenic
cucumber using CRISPR/Cas9 technology
(Chandrasekaran et al., 2016)
Objective: To disrupt the function of the recessive eIF4E (eukaryotic translation initiation
factor 4E) gene through gene knockout in cucumber by the CRISPR/Cas9 system
30

31. (Chandrasekaran et al., 2016)
Materials and methods:
• eIF4E is a plant cellular translation factor essential for the Potyviridae life cycle, and natural point mutations in
this gene can confer resistance to potyviruses
• In cucumber, two eIF4E genes have been identified, eIF4E (accession no. XM_004147349) (236 amino acids)
and eIF(iso)4E (accession no. XM_004147116.2) (204 amino acids), which share 56% nucleotide and 60%
amino acid homology.
• Agrobacterium mediated transformation
31

32. (Chandrasekaran et al., 2016)
(A) Schematic representation of the
cucumber eIF4E genomic map and the
sgRNA1 and sgRNA2 target sites
(B) Restriction analysis of T0 polymerase
chain reaction (PCR) fragments of
CEC‐1, CEC1‐4 and CEC2‐5.
(C) Alignment of nine colony sequences
from the undigested fragment of line 1
with the wild‐type (wt) genome
sequence.
Figure 1: Gene editing of eIF4E mediated by CRISPR/Cas9 in transgenic cucumber plants 32

33. (Chandrasekaran et al., 2016)
(A) PCR restriction analysis of
Cas9/sgRNA1‐mediated mutations (top panel)
and transgene insertion (bottom panel) in 10
representative T1 cucumber plants and
non‐mutant wild‐type (wt).
(B) Alignment of four representative eif4e mutant
plants with the wild‐type sequence.
Figure 2: Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1‐1 line 33

34. (A)PCR restriction analysis of
Cas9/sgRNA1‐mediated mutations (top
panel) and transgene insertion (bottom
panel) in eight T1 cucumber plants and
non‐mutant wild‐type (wt).
(B) Alignment of three eif4e transgenic
mutant plants 4, 5 and 6 with the
wild‐type sequence.
Figure 3: Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1‐4 line
(Chandrasekaran et al., 2016)
34

35. (Chandrasekaran et al., 2016)
(A) PCR restriction analysis of
Cas9/sgRNA2‐mediated mutations (top
panel) and the presence of the
Cas9/sgRNA2 transgene (bottom panel) in
eight representative T1 cucumber plants.
(B) Alignment of four
representative eif4e mutant plants with the
wild‐type sequence.
Figure 4: Genotyping of the Cas9/sgRNA2‐mediated mutation in T1 progeny plants of the CEC2‐5 line 35

36. (Chandrasekaran et al., 2016)
(A)Disease symptoms (leaves and
plants) of heterozygous (Het‐mut),
homozygous (Hom‐mut) and
non‐inoculated (Control) plants of
the CEC1‐1‐7‐1 T3 generation at
10 days post‐infection (dpi).
(B) RT‐PCR analysis at 14 dpi in
homozygous eif4e mutant plants
Figure 5: Homozygous eif4e mutant plants exhibited immunity
to Cucumber vein yellowing virus (CVYV) infection
36

37. Table 1. Response of T3 generation plants of non‐transgenic CEC‐1‐7‐1 and CEC2‐5‐M‐4n lines to Cucumber vein
yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV), Papaya ring spot mosaic
virus‐W (PRSV‐W), Cucumber mosaic cucumovirus (CMV) and Cucumber green mottle mosaic tobamovirus (CGMMV)
infection at different days post‐infection (dpi).
(Chandrasekaran et al., 2016)
37

38. (Chandrasekaran et al., 2016)
(A) Disease symptoms of heterozygous (Het‐mut),
homozygous (Hom‐mut) and non‐inoculated
(Control) plants of the CEC1‐1‐7‐1 T3 generation
at 25 days post‐infection (dpi).
(B) RT‐PCR analysis in homozygous eif4e mutant
plants (1–10), heterozygous plants (Het‐mut) and
non‐inoculated plant (H) at 14 dpi.
(C) Relative (real‐time quantitative RT‐PCR) ZYMV
RNA accumulation in CEC1‐1‐7‐1 heterozygous
(Het‐mut) and two classes of homozygous
mutant: resistant (Resistant) and breaking
(Break).
Figure 6: Homozygous eif4e mutant plants exhibited resistance
to Zucchini yellow mosaic virus (ZYMV) infection
38

39. (A) Disease symptoms of heterozygous (Het‐mut),
homozygous (Hom‐mut) and non‐inoculated
(Control) plants of CEC1‐1‐7‐1 T3 generation at
21 days post‐infection (dpi).
(B) (RT‐PCR) analysis of PRSV‐W RNA
accumulation in homozygous plants (1–8),
heterozygous plant (Het.) and non‐inoculated
plant (H) at 14 dpi.
(C) Relative (real‐time quantitative RT‐PCR)
accumulation of PRSV‐W RNA in CEC2‐5‐M‐9
heterozygous (Het‐mut) and three classes of
homozygous mutant: resistant (Resistant),
breaking (Break) and recovering (Recovery).
Figure 7: Homozygous eif4e mutants exhibited resistance to Papaya ring spot mosaic virus‐W (PRSV‐W)infection
(Chandrasekaran et al., 2016)
39

40. Conclusion:
40
• Disruption of the eIF4E gene in cucumber by CRISPR/Cas9 sgRNA led to the development of
virus‐resistant plants without otherwise affecting the plant genome.
• Three generations of backcrossing produced virus‐resistant plants free of genetic modification, and
thus would be considered safe for human consumption and for release into the environment.
• Homozygous mutants are showing virus resistance whereas heterozygous mutants are highly
susceptible and also observed the breaking of resistance by accumulation of RNA through RT-PCR.

41. Plant species Virus Target gene Gene function Strategy Reference
Nicotiana
benthamiana and
Arabidopsis thaliana
BeYDV CP, Rep and
IR
RCA
mechanism
Agrobacterium-mediated transformation of leaves with
Cas9/gRNA expression plasmid vectors
Ji et al., 2015
Nicotiana
benthamiana
BSCTV LIR and
Rep/RepA
RCA
mechanism
Agrobacterium-mediated transformation of leaves with
Cas9/gRNA expression plasmid vectors
Baltes et al., 2015
Nicotiana
benthamiana
TYLCV
BCTV MeMV
CP, Rep and
IR
RCA
mechanism
Agrobacterium-mediated transformation of leaves with a TRV
vector in Cas9 overexpressing plants
Ali et al., 2015
Nicotiana
benthamiana
CLCuKoV
MeMV
TYLCV
CP, Rep and
IR
RCA
mechanism
Agrobacterium-mediated transformation of leaves with a TRV
vector in Cas9 overexpressing plants
Ali et al., 2016
Nicotiana
benthamiana
TuMV GFP1, GFP2,
HC-Pro, CP
Replication
mechanism
Agrobacterium-mediated transformation of leaves with a TRV
vector in Cas13a overexpressing plants
Aman et al., 2018
Nicotiana
benthamiana and
Arabidopsis thaliana
CMV TMV ORF1, 2, 3,
CP and
3’UTR
Replication
mechanism
Agrobacterium-mediated transformation of leaves with
FnCas9/gRNA expression binary vectors Floral dipping for
Arabidospsis
Zhang et al., 2018
Cucumis sativus CVYV ZYMV
PRSV-W
eIF4E Host factor for
RNA viruses
Translation
Agrobacterium-mediated transformation of cut cotyledons
(without embryo) with Cas9/gRNA binary vectors
Chandrasekara et
al., 2016
Arabidopsis thaliana TuMV eIF(iso)4E Host factor for
RNA viruses
Translation
Agrobacterium-mediated transformation with Cas9/gRNA
recombinant plasmid binary vectors (floral dipping)
Pyott et al., 2016
Oryza sativa L.
japonica
RTSV eIF4G Host factor for
RNA viruses
Translation
Agrobacterium-mediated transformation of immature embryos
with Cas9/gRNA expression plasmid vectors
Macovei et al.,
2018
CRISPR/Cas9 applications for plant virus resistance.
41

42. Engineering canker-resistant plants through CRISPR/Cas9-targeted
editing of the susceptibility gene CsLOB1 promoter in citrus
Aihong Peng, Shanchun Chen, Tiangang Lei, Lanzhen Xu, Yongrui
He, Liu Wu , Lixiao Yao and Xiuping Zou
Objective:
• To provide an efficient approach for generation of canker-resistant cultivars through modification of the
CsLOB1 promoter in citrus.
• To improve citrus canker resistance via promoter-targeted modification of the susceptibility gene CsLOB1
in Wanjincheng orange (Citrus sinensis Osbeck)
(Peng et al., 2017)
42

43. Materials and Methods
43
• Wanjincheng orange plants were obtained from the National Citrus Germplasm Repository, Chongqing,
China.
• EBEPthA4 codon enhances the CsLOB1 promoter
• in vitro assay for disease resistance of mutant plants to Xanthomonas citri subsp. citri by Pinprick
inoculation
• Gene expression analysis through qRT-PCR
(Peng et al., 2017)

44. (Peng et al., 2017)
Figure 1: CRISPR/Cas9-mediated modification of the CsLOB1
promoter in Wanjincheng orange (Citrus sinensis Osbeck)
(a) Schematic structure of CsLOB1. CsLOB1
contains two exons indicated by gray
rectangles.
(b) Schematic diagram of pCas9/CsLOB1
sgRNA vectors.
(c) Representative chromatograms of CsLOB1
promoter mutations.
44

45. Table 1: Proportions of mutant genotypes obtained with five
sgRNAs in Wanjincheng orange (Citrus sinensis Osbeck)
(Peng et al., 2017)
45

46. Table 2: Proportions of mutation types obtained with five
sgRNAs in Wanjincheng orange (Citrus sinensis Osbeck)
(Peng et al., 2017)
46

47. (Peng et al., 2017)
Figure 2: Expression characteristics of CsLOB1 in
Wanjincheng orange mutants
(a) Expression of CsLOB1 in mutant plants after
Xanthomonas citri subsp. citri (Xcc) inoculation.
(b) Time-course of CsLOB1 expression in mutants after
Xcc inoculation.
(c) Statistical analysis of transcripts of CsLOB1G and
CsLOB1− in citrus mutants.
At 5 dpi, CsLOB1 cDNA from infected leaves was
amplified by PCR, cloned into the pGEM® -T Easy
vector, and sequenced
47

48. (Peng et al., 2017)
Figure 3: Identification of citrus canker resistance in Wanjincheng orange mutants
(a) Representative sequences of
CsLOB1 mutations induced by
CRISPR/Cas9.
(b) (b, c and d) Assay of resistance to
Xanthomonas citri subsp. citri
(Xcc) in mutant plants.
Fully expanded leaves of mutant
lines and the wild type were treated
with 105 CFU ml−1 Xcc.
48

49. (Peng et al., 2017)
Disease lesion area (c) and disease index
(d) of leaves of each mutation line were
investigated at 9 dpi.
(e) Growth of Xcc in leaves of mutant
plants.
49
Figure 3: Identification of citrus canker resistance in
Wanjincheng orange mutants

50. Figure 4: In vivo assay of citrus canker resistance in Wanjincheng orange mutants
Leaves were infiltrated with Xanthomonas citri subsp.
citri (Xcc) suspensions.
• At 6 dpi, Pustules were detected in wild type, but
absent or significantly reduced in mutant plants.
• At 12 dpi, severe canker symptoms were detected in
wild type whereas markedly reduced symptoms were
observed in S2-5 and S2-12. No canker symptoms
were found in S2-6 and S5-13.
(Peng et al., 2017)
50

51. Conclusion:
• Deletion of the entire EBEPthA4 sequence from both CsLOB1 alleles conferred the highest level of resistance to citrus
canker
• 42.0% of the mutant plants harbored the desired modifications and 23.5% of these mutants showed resistance to citrus
canker.
• S2-12 and S2-5 showed that mutation of CsLOB1G alone is sufficient to enhance citrus canker resistance, which indicated
that CsLOB1G is a dominant allele in TAL-induced Xcc virulence in Wanjincheng orange.
• The S5-13 chimera mutant showed a high level of resistance and no citrus canker symptoms, although only 32.4% of the
modified EBEPthA4 was present in the CsLOB1 promoter speculate that this mutation occurred possibly in a specific cell
layer, such as the L1 cell layer (early barrier to pathogen infection).
51
(Peng et al., 2017)

52. CRISPR/Cas9 applications for plant bacterial resistance
Plant
species
Bacteria Target
gene
Gene function Strategy Reference
Oryza
sativa
Bacterial blight
(Xanthomonas
oryzae pv. oryzae)
SWEET13 Sucrose transporter
gene
Agrobacterium-mediated
transformation of embryogenic
callus with Cas9/gRNA
expression plasmid vectors and
TALEN
Zhou et al.,
2015; Li et al.,
2012
Citrus
paradisi
Citrus canker
(Xanthomonas citri
subspecies citric)
LOB1 Susceptibility (S)
gene promoting
pathogen growth
and pustule
formation
Agrobacterium-mediated
transformation of epicotyl with
Cas9/gRNA expression plasmid
vectors
Jia et al., 2016
Citrus
sinensis
Osbeck
Citrus canker
(Xanthomonas citri
subspecies citric)
LOB1 Susceptibility (S)
gene promoting
pathogen growth
and pustule
formation
Agrobacterium-mediated
transformation of epicotyl with
Cas9/gRNA expression plasmid
vectors
Peng et al.,
2017
Malus
domestica
Fire blight (Erwinia
amylovora)
DIPM-1
DIPM-2
DIPM-4
Susceptibility
factor involved in
fire blight disease
PEG-mediated protoplast
transformation with CRISPR
ribonucleoproteins
Malnoy et al.,
2016
52

53. Advantages of CRISPR/Cas9:
• The introduced mutations are inherited by the next generation
of plants, indicating that plant genome editing can be used for
plant research and the production of useful plants.
• An important advantage of using the CRISPR/Cas9 system is
the possibility of simultaneously editing multiple target genes
• Simultaneous targeting of multiple sites also can induce
deletions with defined sizes between target sites
• Gene stacking( Simultaneous breeding for multiple diseases)
Limitations:
• off-target effects, i.e., unintended mutations at unintended
sites induced by genome editing.
53

54. (Haque et al., 2018)
Examples of CRISPR/Cas9-mediated genome editing in crop plants cultivated in the tropical
climates for development of tolerance to abiotic and biotic stresses.
54

55. (Haque et al., 2018)
55

56. 56
❖ This novel technology CRISPR/Cas 9 has the potential to expedite the development of pest resistance in many
crops without the need for extensive backcrossing and genetic manipulation with wild sources of resistance.
❖ It has the multiple uses in genome manipulation i. e., gene repression, Suppression of the gene promoter, gene
inactivation etc., through the knockout or knockdown of the gene.
❖ Requires less time in introducing a resistant variety and also doesn’t harm the environment.
Conclusion:

57. QQ
Queries??
57