CRISPR is a novel genome editing tool using Cas9 nuclease guided by CRISPR RNA. The document discusses CRISPR's mechanism and applications in editing plant fungal pathogens. It provides examples where CRISPR was used to modify genes conferring disease resistance in rice, citrus, and viruses. The advantages of CRISPR include specificity, minimizing off-target effects, and applicability across species. Challenges include off-target effects and mosaicism. Highlights of studies editing fungi include creating mutants in Alternaria alternata and Ganoderma lucidum. CRISPR also suppressed pathogenicity in Sclerotinia sclerotiorum by editing oxalate genes.

1. WELC0ME
GOOD MORNING TO
ALL 1

2. Crispr – a novel tool for
genome editing
2
k.Vignesh ,
M.Sc (Ag) –II nd Year,
Deptof plantpathology

3. 3
A CRISPR array is composed of a series of repeats interspaced by
spacer sequences acquired from invading genomes.
(Gitaitis et al.1985)
CRISPR– Clustered RegularlyInterspaced ShortPalindromic Repeats

4. 4
Why genome editing?
(Mao Y et al.2015)
Nucleases such as CRISPR can cut any targeted
position in the genome and introduce a
modification of the endogenous sequences for
genes that are impossible to specifically target
using conventional RNAi.

5. DISEASE MANAGEMENT METHODS
 Cultural Methods
 Mechanical Methods
 Chemical Methods
 Physical Methods
 Biological Methods
(Melotto et al. 2008)

6. CULTURAL METHODS
 May suppress some pathogens, but increase others.
 May require community wide adoption.
 Generally Slower than Chemicals for Controlling out breaks.
 Timing decides Success.
 No Complete Control
 Required Long term planning.
DEMERITS
(Melotto et al.2017)

7. CHEMICAL METHODS :
 Chemical May be Non Specific and kills
beneficial Microorganism.
 Pathogen may develop resistance to the
Fungicides, Antibiotics.
 Chemicals May enter the food chains and harm
other organisms.
7
DEMERITS
(Melotto et al.2017)

8. BIOLOGICAL METHODS
 A Natural enemy may also damage the crop,
especially when large numbers are needed to control
pathogen.
 It is a slow process its take a lot of time.
 When introduce is new species to the environment,
there is a risk of disrupting the natural food chain.
8
DEMERITS
(Schneider et al. 1977)

9. PHYSICAL METHODS
• Requires a limited infested area and limited
population.
• Requires Public information & sensibilisation.
• Special Authorization.
9
DEMERITS
(Schneider et al.1977)

10. Other GENOME EDITING TECHNIque
GMO – GENETICALLY MODIFIED ORGANISM :
(Du et al.2014)
10
Genetically modified organisms are living organisms whose
genetic material has been artificially manipulated in a laboratory
through genetic engineering. This creates combinations of plant,
animal, bacteria & virus genes that do not occur in nature or through
traditional crossbreeding methods.

11. 11
(Robert-Seilaniantz et al. 2011)
TRANSGENIC PLANTS
 Modification of DNA using
genetic engineering techniques.
Aim is to introduce a new trait to
the plant. The inserted sequence
is known as transgene.

12. 12
NATURAL GENE EDITING
(Robert-Seilaniantz et al. 2011)
Gene editing is a type of genetic engineering
in which DNA is inserted deleted, modified or
replaced in the genome of a living organism.

13.  CRISPR(/’KRISPER) is a family of DNA sequences in bacteria.
 A palindromic repeat, the sequence of nucleotides is the same in both
directions.
 Each repetition is followed by short segments of spacer DNA from
previous exposures to foreign DNA (e.g., A virus or plasmid).
 Small clusters of cas (CRISPR-associated system) genes are located next to
CRISPR sequences.
 The CRISPR/cas system is a prokaryotic immune system .
CRISPR – Clustered Regularly Interspaced Short Palindromic Repeats
(Virginia M.G. Borrelli et al. 2016)13
INTRODUCTION

14. MAINACTORSIN CRISPR/CAS9 PATENT WAR*
14
Emanuelle charpenter
Feng zang
JenniferDoudna
(Joginder Pal et al.2017)

15. TIMELINE
SI. No Year CONTRIBUTION
1. 1987 First record of CRISPR cluster repeats was reported in Escherichia Coli
(Ishino et al. 1987) [12]
2. 2008 CRSIPR system can act upon specified DNA targets; Spacers are
converted into mature crRNAs which acts as a small guide RNAs (Brouns
et al. 2008).
3. 2010 Cas9 is directed by spacer sequences and cleaves target DNA via Double
strands breaks (Garneau at al.2010).
4. 2016 Development of resistance to a ssRNA plant virus through CRISPR/Cas 9
deal mutagenesis of host gene elF(iso) 4E that codes a protein which is
necessary for virus replication and multiplication of same virus (Pyott et
al. 2016)
(Joginder Pal et al.2017)

16. MODE OF ACTION OF CRISPR
16
• The CRISPR immune
system works to protect
bacteria from repeated
viral attack via three
basic steps:
• Adoption
• Expression of cr RNA,
• Interference
(Jingtao Li et al.2015)

17. GENERAL PROTOCOL
1. SELECT GENOMIC TARGET
a) 20 bp sequence followed by the PAM (NGG)
b) Use online tools to minimize off – targeting.
2. DESIGN sgRNA
a) sgRNA is expressed using a small RNA promoter, such as U6p or U3p
b) Guide sequence should match the target, except for the first nucleotide (5’ G or A)
that does not have to match.
3. ASSEMBLE CAS9/ sgRNA CONSTRUCT
4.DELIVER TO PLANTS
 Protoplast transformation
 Agrobacterium transformation
 Callus bombardment
5.REGENERATE AND SCREEN TRANSGENIC PLANTS FOR GENE EDITING
EVENTS
(Alessandra et al. 2016)

18. The Basicflow of CRISPR/ Cas 9 editing of
target genes
18
(Jingtao Li et al.2015)

19. 19
(Chini et al.2007)
tOOLS of Crispr

20. terminology
PAM – ( Protospacer adjacent Modify)
 A 2-6 base pair DNA sequence immediately following the
DNA sequence targeted by the case 9 nucleus in the
(CRISPR bacterial adaptive immune system. PAM is a
component of the invading virus or plasmid, but is not a
component of the bacterial CRISPR locus.
20
(Ishiga et al. 2013)

21.  (ZNFS) were the first of the “Genome Editing” nucleus to
hit the sense. ZFN consists of a zinc finger DNA binding
domain and DNA cleavage domain of FOK-1, the most
thoroughly studied type IIS restriction endonucleaus.
FOK 1 – Flauobacterium okeanokoites
(Ishiga et al. 2013) 21
ZFN – (Zincfinger Proteins)
HighlySpecificGenomicScissors

22. CR RNA –CRISPR RNAS
Guide to degrade the involving nucleic acid (cas 9)
 CRISPR RNAs (crRNAs) are transcripted from this CRISPR locus.
The crRNAs are then incorporated in the effector complex's, where the
crRNA guides the complex to the involving nucleic acid and the CAS
proteins degrade this nucleic acid.
(Ishiga et al. 2013)

23. TRACRRNA – TRANS ACTIVATING
CR RNA
RNA Specific ribonuclease to formcrRNA/ TracrRNA whichplace a rolein the
maturationof crRNA.
The tracr RNA or trans activating cr RNA is made of up of a
longer stretch of bases that are constant and provide the “Stem
Loop” structure bound by the CRISPR nucleus.
(Chini et al.2007)

24.  TALEN are restriction enzymes that can be engineered
to cut specific sequences of DNA. They are made by fusing a
TAL effector DNA – binding domain to a DNA cleavage
domain (a nucleus which cuts DNA strands).
TALEN – TranscriptionActivator –
like effector nucleases
(Ishiga et al. 2013)

25. 25
(Kim et al. 2010)
MILESTONES OF CRISPR/CAS9 IN CROP
BIOTECHNOLOGY

26. 26
(Kim et al. 2010)
Basic flowchart of the CRISPR/Cas9 genome editing system

27. 27
Advantages of
CRISPR/Cas9
tehnology
1.Site specfic
mutagenesis 2. Minimizing off-
target mutations
3.Applicablility
across awide range
of organisms with
multifuction
4.Multiplexing
5.Efficient and
easy to use
6.Inexpensive and
less time
consuming
7.Multifunctional
programmability;
Delete, insert or
repair and cloning
is not necessay
8.Beyond genome
editing
(Joginder pal et al. 2019)

28. Plant Fungus Classification,
Family
CRISPR/ Cas System edited
region of Plant genome
Oryza sativa Magnaporthe
oryzae
Ascomycota/
Sordariomycetes
CRISPR/ SP Cas 9. Modification
of the OsERF922 gene
Bacterium
Citrus sinesis
osbeck
Xanthomonas
citri
Proteobacteria/
Gammaproteo-
bacteria
CRISPR/ SP Cas 9. Modification
of the CsLOB1/ gene Promoter
Virus
Nicotiana
benthamiana,
Arabidopsis
thaliana
Beet severe curly
top virus
(BSCTV), DNA
– Containing
Virus.
Gemiiviridae CRISPR/SpCas9. sgRNAs
targeting regions within coding
and non-coding sequences of
viral genome.
( S.S Makarova et al.2017)
Examples of pathogen resistant plants generated using the CRISPR/Cas 9 technology

29. 29

30. (Syed Shan-e-Ali Zaidi et al.2018)

31. Gemini
Virus
Ss
DNA
ds
DNA
RCR
4 Virus starts
replicang
1 Cas9 and
gRNA are
transcribed
2 cas9
binds
with gRNA
3
Virus
infects
the
plant
cell
Gemini
virus
gRNA
Cas 9
Chopped virus
indel
Plant
genome
6 Virus
replication is
interrupted
5 Cas9-
gRNA
Complex
binds with
virus
(Syed Shan et al.2018)
Plant Cell

32. S. No Fungal species Edited gene References
1 Alternaria alternata Polyketide synthase A(pksA)
1,3,8-THN reductase(bmr2)
Wenderoth et al., 2017
2 Aspergillus aculeatus Polyketide synthase (albA)
Laccase (yA)
Nodvig et al., 2015
3 Ganoderma lucidum
Ganoderma lingzhi
Orotate phospho ribosyl transferase
(ura3)
Qin et al., 2017
4 Pyricularia oryzae Scytalone dehydratase (sdh)
Suppressor of RAD six (sdr2)
Arazoe et al., 2015
5 Sclerotium sclerotiorum Oxaloacetate acetylhydrolase
(Ssoah1)
Polyketide synthase (Sspks13)
Li et al., 2018
6 Ustilago maydis Central regulator of pathogenic
development (bW2) (bE1)
Schuster et al., 2016
32
(Munoz et al.)
Applicationof CRISPR/CAS9 FORGENESILENCINGIN
FILAMENTOUSFUNGI

33. 33

34.  First description of CRISPR/Cas9 application in A. alternata establishment of a pyrG
mutant by use of GFP
 A. alternata lacks a sexual cycle and classical genetic approaches cannot be combined
with molecular biological methods.
 Gene deletions often result in hetero karyotic strains and gene function analyses are
rather tedious
 We have used the tool to inactivate two genes of the melanin biosynthesis pathway and
in addition created a pyrG auxotrophic mutant. Two genes of the melanin biosynthesis
pathway, pksA and bmr2, were chosen as targets. Several white mutants were obtained
after several rounds of strain purification through protoplast regeneration or spore
inoculation. Mutation of the genes was due to deletions from 1 bp to 1.5 kbp.
 The CRISPR/Cas9 system was also used to inactivate the orotidine-5-phosphate
decarboxylase gene pyrG to create a uracil-auxotrophic strain. The strain was counter-
selected with fluor-orotic acid and could be re-transformed with pyrG from Aspergillus
fumigatus and pyr-4 from Neurospora crassa.
 In order to test the functioning of GFP, the fluorescent protein was fused to a nuclear
localization signal derived from the StuA transcription factor of Aspergillus nidulans.
 After transformation bright nuclei were visible.
(Maximilian Wenderoth et al.)
highlights

35. (Maximilian Wenderoth et al.)
Hoechst GFP Merge
35

36. 36

37.  CRISPR-Cas9 assisted gene disruption was
demonstrated for the first time in higher fungi.
 The ura3 gene of Ganoderma lucidum was disrupted
by the codon-optimized Cas9 and in vitro transcribed
gRNA.
 This work may help to provide a widely applicable
approach of gene disruption in higher fungi.
37
(Hao qin et al. 2017)
highlights

38. (Hao qin et al. 2017)

39. (Hao qin et al. 2017)

40. 40

41.  The oxalic acid is induce the pathogenity of
Sclerotinia sclerotiorum.
 The CRISPR/Cas 9 system supress the pathogenicity
effect of S. sclerotiorum .
 CRISPR/Cas 9 edited genome Oxaloacetate acetyl
hydrolase(Ssoah 1).
(Hongyu Pan et al.2018)
highlights

42. (Hongyu Pan et al.2018)

43. (Hongyu Pan et al.2018)

44. DISADVANTAGES
OFF SITE EFFECTS
MOSAICISM :
MULTIPLE ALLELES
 Target sequences may be limited due to PAM
sequences.
 Necessity of knowing gene function and
sequences.
44
(Luca malfatti et al. 2019)

45. 45

46. THANKYOU