this presentation file is based on a article, which discuss about the application and advancement of CRISPR-Cas9 in crop plants.

1. RNA-guided genome editing tool CRISPR-Cas9:Its
Applications and Achievements In Model and Crop Plants
Om Prakash Patidar, Chirag Gautam, Girish Tantuway,
Sunil Kumar, Ashok Yadav , Dharam Singh Meena and Arvind Nagar
JOURNAL OF PURE AND APPLIED MICROBIOLOGY, Dec. 2016.
Presented By-
Sabiha Sultana
Parmita Mandal
Munia Akter
Department of Biotechnology and Genetic Engineering
Noakhali Science and Technology University
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2. Outline
Introduction CRISPR/Cas9 History
Type and
components
Mechanism
of action
Application
on plants
Considerations
and advances
Conclusion
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3. Introduction
• Sequence specific and efficient genome editing tool used to
make changes in genetic material
• Meganucleases, ZFNs, TALENs and CRISPR-Cas9 nuclease
are some genome editing tools
• Other than CRISPR-Cas9 nuclease, all other genome editing
tools have some limitations which reduces their utilization
• CRISPR-Cas9 nuclease overcomes the limitations of other
tools
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4. • The ease of cas9 targeting, efficient site specificity and
ability for multiple editing makes CRISPR-Cas9 more useful
in genome editing
• Now we will discuss about CRISPRs recent developments
and applications in model and field crop plants
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5. CRISPR/Cas9
• CRISPR- Clustered Regularly Interspaced Short
Palindromic Repeats
• Cas (CRISPR- associated) proteins can target and
cleave invading DNA in a sequence – specific
manner
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6. History
1987
• Researchers find CRISPR sequences in Escherichia coli, but do not
characterize their function
2000
• Present in more than 40% of sequenced bacteria and 90% of archea
2005
• Systemic analysis of the spacer sequences separated the individual
direct repeats
2011
• Types and components of CRISPR were known and efforts to apply it
for genome editing began
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7. Types and Components
• Three types: Type I, II and III
• Type II is highly used.
• CRISPR system consists of: crRNA, tracrRNA and cas9 nuclease
protein
• SgRNA is constructed through fusion of crRNA and tracrRNA.
• PAM sequence is present at 3’ end of target sequence
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8. Mechanism of action
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• Figure: Mechanism of action of RNA guided CRISPRCas9 in creation of Double Strand
Breaks (DSB) in the target DNA sequence and the possible pathways of DNA repair.

9. Application on plants
• Rice blast is one of the most destructive diseases affecting rice
• Improvement of rice blast resistance by engineering a CRISPR/Cas9
SSN (C-ERF922) targeting the transcription factor gene OsERF922 in
rice
• Mutant plants harboring the desired gene modification but not
containing the transferred DNA were obtained by segregation in the
T1 and T2 generations
• In T2 generation homozygous mutants on comparison with wild type,
it was found that lesions for blast resistance reduced significantly and
other desirable agronomic traits remained same
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10. Figure: Identification of blast resistance in homozygous mutant rice lines.
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a b
c
Fujun Wang et al

11. CRISPR cure powdery mildew in wheat
• In wheat, powdery mildew is caused by Blumeria graminis f. sp. tritici
(Bgt), which is one of the most destructive plant pathogens
worldwide
• Use of TALEN and CRISPR/Cas9 technologies to target and successfully
knock out the three homeoallelic genes of the mildew-resistance
locus (MLO) in wheat to generate plants resistant to powdery mildew
disease
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12. Figure: (a) NHEJ-mediated knock-in of a GFP reporter gene at a TaMLO site in wheat protoplasts.
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Yanpeng Wang et al

13. (b) Measurement of GFP knock-in efficiency in wheat protoplasts by flow cytometry.
(c) Sequencing of 5′ and 3′ junctions confirm NHEJ-mediated knock-in events.
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Yanpeng Wang1,3

14. Resist Browning in White Button Mushroom
• CRISPR-Cas9 engineered the common white button mushroom
(Agaricus bisporus) fungus to resist browning
• Achieved by targeting the family of genes that encodes polyphenol
oxidase (PPO), an enzyme that causes browning
• Knocked out one of six PPO genes by deleting a few of base pairs in
the mushroom’s genome and reduce the enzyme’s activity by 30%
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15. Application on plants
• Simple and efficient genome-editing approach to regenerate
mutant plants from wheat callus cells done by TECCDNA, or
through TECCRNA
• Homozygous mutant plants with no detectable transgenes are
identified in T0 populations.
• CRISPR/Cas system was coupled with VIGS (Virus induces gene
silencing) technique in tobacco
• The cas9/ single guide RNA (sgrna) system, have emerged as
potent new tools for targeted gene knockout in bacteria, yeast,
fruit fly, zebrafish and human cells
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16. Consideration & Recent advances
• Factors like cas9/sgRNA constructs or delivery methods effects its
efficiency of causing mutation
• Improper concentration ratio between cas9 and sgRNA leads to off-
target activity.
• Codon optimization of cas9 leads to efficient genome editing
• Optimal promoters play role in expressing Cas9 protein inside the cell.
• In dicots-35S CaMV for Cas9 and U6 for SgRNA expression.
• In monocots- 35S and Ubi for Cas9 expression
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17. • With the development of dCas9 (dead Cas9) accuracy is increased by
5000 fold in which Cas9 nuclease activity is mutated and attached
with FokI
• CRISPaint allows precise and efficient integration of large
heterologous DNA cassettes into eukaryotic genome
• use the CRISPR/Cas9 system to introduce a double-strand break
(DSB) at a user defined genomic location
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18. CONCLUSIONS
• Enhancing blast resistance in rice
• Bringing powdery mildew resistance in wheat
• Not only for genome editing purposes but also for other gene
expression regulation and epigenetic modifications
• Overcomes the traditional limitations of genetically modified crops
• Off-target mutations, epigenetic effects on genome editing, effects of
nearby genes
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19. Thank you
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