This document discusses the use of CRISPR Cas technology in plant disease management. It begins with an introduction to CRISPR Cas technology, describing how it was originally discovered as a prokaryotic antiviral defense system. It then discusses how CRISPR Cas technology works and its various components. The majority of the document focuses on examples of how CRISPR Cas technology has been used to manage bacterial, fungal, and viral diseases in various crop plants by targeting genes involved in disease susceptibility or pathogenesis. It concludes by discussing some advantages and challenges of CRISPR Cas technology and its future applications in advanced crop breeding and plant pathology.

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Dept. of Plant Pathology
WELCOME

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Crops are susceptible to a larger set of pathogens
(fungi, bacteria,viruses etc.) causing severe
economic loss
(Nazarov et al., 2020)
Importance of plant diseases

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Crop loss by plant pathogens
(Khan and Sharma, 2020)

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Conventional breeding alone will not bridge the gap between
current level of crop production and expected levels in the
decades to come in the food production systems
(Bhattacharya et al., 2021)

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Genetic improvement is helpful to minimize
chemicals use for crop protection offers a promising
alternative without a direct effect on living things
and the environment
(Pohare et al., 2021)

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DNA and Gene
Character
Expression
(Mushtaq et al., 2019)

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What is genome ?
A genome is the total genetic material of an organism
A genome sequence - (A, C, G, and T for DNA genomes)
- make up all the chromosomes of an individual
(Andolfo et al., 2016)

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Genome editing
Genome editing, or genome engineering, or gene
editing, is a type of genetic engineering in which DNA is
inserted, deleted, modified or replaced in the genome of a
living organism
(Andolfo et al., 2016)

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Different methods of genome editing
Meganuclease
ZFN (Zinc-finger nucleases)
TALEN(Transcription
activator-like effector
nucleases)
CRISPR / Cas technology
(Grohmann et al., 2019)

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Dept. of Plant Pathology
CRISPR Cas technology in plant disease
management
Arun A.T.
2020-21-011
Dept. of Plant Pathology

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1. Introduction
2. What is CRISPR Cas technology?
3. Mechanism of CRISPR Cas technology
4. Management of bacterial diseases
5. Management of fungal diseases
6. Management of viral diseases
7. Problems associated with CRISPR Cas technology
8. Conclusion
9. Future perspective
Contents

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What is CRISPR Cas technology ?
CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats) is a family of DNA sequences
found in the genomes of prokaryotic organisms such as
bacteria and archaea
(Mushtaq et al., 2019)

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CRISPR - first identified in E. coli in 1987 by a Japanese
scientist, Yoshizumi Ishino
Discovery of CRISPR
(El-Mounadi et al., 2020)

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CRISPR sequences play a key role in the antiviral (anti-
phage) defense system of prokaryotes and provide a
form of acquired immunity
(El-Mounadi et al., 2020)

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CRISPR sequences are derived from DNA fragments
of bacteriophages that had previously infected the
prokaryote
They are used to detect and destroy DNA from similar
bacteriophages during subsequent infections
(El-Mounadi et al., 2020)
How the CRISPR sequences are formed?

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Enzyme that uses CRISPR sequences as a guide to
recognize and cleave specific strands of DNA that are
complementary to the CRISPR sequence
Cas ("CRISPR-associated protein")
(Zhang et al., 2021)

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Characteristics of different types of
CRISPR/Cas systems involved in plant-
pathogen interactions
CRISPR
system
Cas9 Cas12a Cas12b Cas13 Cas14
Type and
Class
Type II
Class 2
Type V
Class 2
Type V
Class 2
Type VI
Class 2
Type V
Class 2
Substrate dsDNA dsDNA dsDNA RNA ssDNA
(Gosavi et al., 2020)

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Diagram of the CRISPR prokaryotic antiviral
defense mechanism
(El-Mounadi et al., 2020)

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Presence of CRISPR
Sequenced bacterial genomes Sequenced archaea genomes
(Zhang et al., 2021)

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Emmanuelle charpentier and
Jennifer doudna
Discovery of CRISPR Cas Genome editing
technology
(Zhang et al., 2021)

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Mechanism of CRISPR Cas9 technology
DNA Cutting enzyme
Searching and
binding to similar
DNA sequence
(Asmamaw and Zawdie, 2021)

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(Asmamaw and Zawdie, 2021)

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Management of bacterial diseases using
CRISPR Cas technology

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Management of bacterial leaf blight of rice using
CRISPR Cas technology
(Blanvillain‐Baufume et al., 2017)
S gene (Host plant)
•Help in early pathogen
establishment
•Modulation of host defenses
•Pathogen substenance
EBE

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Functional analysis of two Kitaake edited lines
carrying deletions in EBEs (Effector binding
element site)
(Blanvillain‐Baufume et al., 2017)

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In vivo assay of citrus canker resistance
(Peng et al., 2017)
Xanthomonas axonopodis pv. citri
CsLOB1 Promoter

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Disease index of citrus canker in wild and
mutated lines
(Peng et al., 2017)

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CsLOB1 mutations induced by CRISPR/Cas9
(Peng et al., 2017)

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Wild type and dmr6 mutant lines infected with
Xanthomonas gardneri (Xg153) in tomato
(De et al., 2016)

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Wild type and dmr6 mutant lines infected with
Xanthomonas perforans (Xp4b) in tomato
(De et al., 2016)

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Wild type and dmr6 mutant lines infected with
Pseudomonas syringae (DC3000) in tomato
(De et al., 2016)

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Management of fungal diseases using
CRISPR Cas technology

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The SlMlo1 locus was targeted by two sgRNAs
(Nekrasov et al., 2017)
Mildew locus (mlo) in tomato
Oidium neolycopersici

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Leaves of tomato plants inoculated with Oidium
neolycopersici (5 weeks post inoculation)
(Nekrasov et al., 2017)

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Tomelo
variety

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CRISPR Cas9 mediated mutagenesis of DMR6
(De et al., 2016)

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Macroscopic infection phenotypes of wt and the
indicated mlo mutants of powdery mildew of
wheat
Mildew- locus (mlo)
(Wang et al., 2014)

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Disease symptoms of wild-type (wt) and tamlo-
aabbdd mutant plants
(Wang et al., 2014)

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Musa acuminata –
Cavendish banana –
Triploid (AAA)
Fusarium fungus called
tropical race 4 (TR4)

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•Susceptibility genes to rice blast disease, when these genes are
mutated they leads to disease resistance
•Identified putative orthologs of 10 susceptibility genes. They
will modify these genes using CRISPR-Cas9 editing

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Infection assays of papaya fruits by P. palmivora
wild-type (WT) strain and PpalEPIC8 mutants
(Gumtow et al., 2018)
•Papain, a cystein protease- Plant
defense
•Phytophthora palmivora infects-
cystein protease inhibitors
•Five putative inhibitors
•PpalEPIC8- Loses virulence

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Infection assays of papaya fruits by P. palmivora
wild-type (WT) strain and PpalEPIC8 mutants
(Gumtow et al., 2018)

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Management of viral diseases using CRISPR
Cas technology

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The overview of sgRNA-Cas9-based sequence-
specific system for conferring Gemini virus
resistance in plants
(Ji et al., 2015)
Beet severe curly top virus
(BSCTV)

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Transgenic Nicotiana resistance to Beet severe
curly top virus (BSCTV)
(Ji et al., 2015)

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Resistance against Tomato yellow leaf curl virus
(TYLCV) via the CRISPR/Cas9 system in tomato
Rep
CP
(Tashkandi et al., 2018)

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Evaluation of genome-edited and wild type non
edited banana plants for induction of Banana
streak virus (BSV) symptoms
•Musa accuminata
(A genome)
•Musa balbisiana
(B genome)
•Plantain (AAB)
(Tripathi et al., 2019)

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Cas 13: binds and cleave RNA viruses
(Pyott et al., 2016)

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Schematic of the eIF(iso) 4E locus targeted for
editing by CRISPR/Cas9
(Pyott et al., 2016)
Eukaryotic initiation factor
(eIF) – helps in translation
of RNA to protein

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Turnip mosaic virus (TuMV)-Green fluorescent
protein (GFP) inoculated Arabidopsis
(Pyott et al., 2016)
Eukaryotic initiation
factor eIF(iso) 4E
locus

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Advantages of CRISPR Cas technology
Simplicity
Specificity High engineering feasibility
Multiplex genome editing
Low cost
Efficiency
(Khan, 2019)
Easy handling High accuracy

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CRISPR Cas technology research in India

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•Regulation of genome engineering technologies in India -
GMO(genetically modified organisms)- EPA rules 1989 (Rules for
manufacture, use of genetically engineered organisms or cells,
1989)
•The Ministry of Environment, Forest and Climate change -final
authority - State Governments and DBT (Department of
Biotechnology)
(Bhattacharya et al., 2021)

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Agencies responsible for approval of GMO crops
RDAC: rDNAAdvisory Committee
IBSC: Institutional Biosafety Committee
RCGM: Review Committee on Genetic Manipulation
GEAC: Genetic Engineering Appraisal Committee
SBCC: State Biotechnology Coordination Committee
DLC: District Level Committee
(Bhattacharya et al., 2021)

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Problems associated with CRISPR Cas technology
It is not always efficient
Gene drifts
Non-target mutation
(Pineda et al., 2019)

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CRISPR Cas system provide a novel opportunity to explore the
complex area of plant pathogen interactions
CRISPR Cas technology playing an important role in advanced
crop breeding and other functional genomic studies
Developing resistance against plant pathogens using GE by
CRISPR could prove promising approach to conquer the
breeding barrier
Conclusion

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Future perspective
CRISPR/Cas genome editing is growing with speed, it needs to
use properly in such a way that it will be safe and beneficial to
society as well as environment
CRISPR/Cas genome editing is likely to face similar problem
like GM crops depending on how regulators view this
technology, and how they provide it with equal regulatory
consideration

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Thank You