Genome editing with CRISPR/Cas9 technique shown the potential of DNA-Free products

1. TRANSGENE FREE GENOME EDITING
MOHD KYUM
Plant Breeding and Genetics
Punjab Agricultural University
Ludhiana
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2. CONTENTS!
Need of transgenic
Problems (GMO)
Strategy that can eliminate transgenics
Case studies
Future perspectives
Conclusion
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3. What is Transgenic?
 Exotic gene added to a species through rDNA
technology-Transgene
 The organism that is developed after successful
transformation-Transgenic 3

4. Need of transgenic
• Population expansion • Decrease in arable land • Time consuming
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5. APPLICATIONS
OF
TRANSGENICS
 Herbicide Resistant  Quality Improvement
 Stress Resistant  Pest Resistant
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6. So why say no??
• Toxicity
Problems of GMO
• Marker genes
• Horizontal
Gene transfer 6

7. Contd….
• Super Weeds • Fitness ( G X E )
• Resistance
7

8. Genome Editing
Strategies that can eliminate transgenic
 Specific
location
 Specific type of
sequence
 Specific final
alteration 8

9. GE vs GMO
● Introducing a gene
from other species
● Almost the same as
the WT
● May be accepted
as non-GMO
Why transgene free editing
9

10. Regulatory Overview of Genome Editing
Product
based
Process
based
GMO GMO free
Disadvantages Advantages
Whether
recombination is
present
 Costly and time consuming
 Block the invention
 Save cost and time
 Offer rapid benefits to
the public
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11. Current status of genome-editing legislation
-Sarah M et al (May, 2020)
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12. Mega-
nuclease
ZFNs TALENs CRISPR
Genome
editing
12

13. Meganucleases
o Recognize and cut large sequences (12-40 bp)
o Highly specific and easy to deliver
o Smallest nuclease (165 amino acids)
o Limitation - difficult to design and screen 13

14. Zinc Finger Nucleases
• First generation engineered endonucleases
• Generated by fusing zinc finger DBD to DNA cleaving domain
• Site specific
• Limitation –laborious and low efficiency
14

15. Transcription-activator-like effector
nuclease (TALEN)
• Fusion of TAL effectors DNA binding RE to DNA
cleavage domain
• Contains non specific endonuclease Fok1
• Specific binding due to 34 amino acid repeat motif
• Limitation –time consuming and cost ineffective 15

16. CRISPR-Mediated Gene Editing
• Target cleavage based on RNA/DNA interaction
• Has the ability to target multiple sequences at once
• Highly efficient and easy to target approach 16

17. Mechanism of Natural CRISPR
17

18. Mechanism of Synthetic CRISPR
18

19. CRISPR- Transgene Free..
19

20. Cont..
20

21. Cont..
21

22. CRISPR Mediated Transgene Free Approach
RNP delivery- pre-assembled gRNA and Cas9 delivery
Particle gun Protoplast transformation
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23. Case studies
23

24. • Transgene free RNP technique was reported for the first time
• Petunia has been suggested as a good system for studying genetics of
phenotypic traits
• Pre-assembled gRNA and Cas9 complex delivered into protoplast for
transient expression
• Mutation detection was done through T7 assay and deep sequencing
Case study-1
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25. RNP mediated mutagenesis in Petunia protoplast
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26. Case study-2
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27. Results
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28. Case study-3
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29. Results of RE digestion
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30. Results - Integration free and analysis
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31. Wild Mutant
• Demonstrated the RNP through DNA as well as pre-assembled
RNP complex
• 4 genomic regions LIG,ALS2, MS26 and MS45 were targeted
• Off targets were reduced in case of RNP delivery in comparison
to DNA delivery method
• Biallelic mutation was observed In case of marker free approach
Case study-4
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32. Comparative Results
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33.  Male fertile tassel of WT-maize  Male sterile tassel of biallelic MS45 mutant
Biallelic mutation result through RNP
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34. • Cpf1-crRNA RNP complexes induced indels at several loci
• Cpf1 induces larger deletions in the target site
• No indels at off target site
• Cpf1 guided by a single crRNA; tracrRNA not required
Case study-5
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35. Results
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36. Comparison of different ways of
CRISPR/Cas delivery
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37. Future perspectives
• Off target effects- efforts should be made towards
reducing the frequency of off target effects
• Integration-free – no integration of DNA into the target
organism
• Advances- increased desirable site directed mutagenesis
through new CRISPR advances
• Delivery- efficient and high-throughput methods of
delivery into plant cells
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38. The CRISPR represents a major shift for
development of transgene free plants.
Less off target effects and high frequency of
mutations.
Eliminate problems of transgenics.
Upcoming challenges in case of GE
Conclusions
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39. 39

40. Any Questions…. ?
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41. Questions….?
1. Can we target the organelle DNA through
CRISPR ?
2. Since genomic DNA is highly complex, how
does RNP reach to the target site ?
3. Through RNP technique can we target
different gene ?
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