2. Genome editing: CRISPR-Cas9 and Its Applications in Plants
Akshay S. Sakhare
Sudhir Kumar
Dhandapani R.
&
Amit Goswami

3. Advantages and Disadvantages
Genome editing
ZFNs
TALENs
Advantages and Disadvantages
CRISPR Cas9

4. CRISPR Cas9

5. Cas9 protein
Doudna and Charpentier (2014)

6. Endogenous cellular DNA Repair machinery

7. History of CRISPR Cas9
1980s
CRISPRs were described Japanese researchers
2005
Spacer sequences are of viral origin
2007
CRISPR Cas mediated adaptive immunity
2008
Mature crRNAs guides Cas proteins
2011
tracrRNA essential for maturation of crRNA by ribonuclease III
2012
dual-RNA–guided DNA endonuclease that uses the tracrRNA:crRNA duplex to direct
DNA cleavage
Anti CRISPR Cas
CRISPR Cpf

8. -Was protein based sequence recognition
How this natural bacterial immunity system was transformed into a genome editing tool
Doudna and Charpentier
crRNA + tracrRNA= gRNA
Genome editing
-Was protein based sequence recognition
- Now in CRISPR Cas9 it is RNA based sequence recognition

9. How to use it for genome editing
Vector construction

10. Adavantages
Applications

11. Case study
Rice is the most important crops in the world feeding about 50% of the world population
Rice Blast caused by filamentous fungus Magnaporthe oryzae is one of the most destructive diseases affecting rice in all rice growing countries and often causes serious damage to global rice production
Enhancing the resistance of rice to M. oryzae has been shown to be the most economical and effective approach for controlling rice blast

12. Plant hormones abscisic acid, salicylic acid, jasmonic acid and ethylene play important roles in the defense response
Plant ethylene responsive factors (ERF), a subfamily of the APETELA2/ethylene response
factor (AP2/ERF) transcription factor super family in plants, are involved in the modulation of multiple stress tolerance and have been implicated in multiple responses to abiotic and biotic stresses
Recent literature suggests that up or down regulation of various ERF family genes resulted
in enhanced resistance to various biotic and abiotic stresses.
Knockdown of expression of the rice ERF gene OsERF922 by RNA interference (RNAi) enhanced rice resistance to M. oryzae, indicating that OsERF922 acts as a negative regulator of blast resistance in rice
Conventional breeding methods takes approximately a decade to pyramid multiple blast
resistance genes into a rice variety via crossing and backcrossing

13. The japonica rice variety Kuiku13 1
The Cas9/sgRNA-expressing vectors (pC-ERF922, pC-ERF922S2) was transformed into an Agrobacterium tumefaciens strain EHA105 by electroporation. Embryogenic calli derived from the japonica rice variety Kuiku131 were transformed with Agrobectrium.
Hygromycin-containing medium was used to select hygromycin-resistant calli, and then the hygromycin-resistant calli were transferred onto regeneration medium for the regeneration of transgenic plants. After 2–3 months of cultivation, transgenic seedlings were transferred to a field during the rice growing season.

14. Nucleotide sequences at the target site (S2) in the 7 T0 mutant rice plants.

15. Pathogen inoculation- 6 homozygous T2 mutant rice lines were inoculated in field
Characterization of agronomic traits
plant height, flag leaf length and width, the number of productive panicles, panicle length, the number of grains per panicle, the seed setting rate, and thousand seed weight after the rice had reached maturity.
Nucleotide sequences at the target site (S2) in the 6 homozygous T2 mutant rice lines

16. Characterization of the blast resistance phenotype of the rice mutants
The mutant rice lines and wild-type plants at the seedling stage
The average area of lesions formed
The average length of lesions formed on the inoculated leaves
The mutant rice lines and the wild-type plants at the tillering stage

17. Analysis of the agronomic traits of 6 homozygous T2 mutant lines

18. Conclusion
Gene modification via CRISPR/Cas9 has successfully enhanced the resistance of rice against M. Oryzae.
Gene modification via CRISPR/Cas9 was a useful approach for enhancing blast resistance in rice.

19. Future perspectives References
Although CRISPR/Cas systems show great promise and flexibility for genetic engineering, sequence requirements within the PAM sequence may constrain some applications.
Directed evolution of the Cas9 protein should offer a path toward PAM independence, and may also provide a means to generate an even more efficient Cas9 endonuclease.
References
J A. Doudna and E. Charpentier (2014). The new frontier of genome engineering with CRISPR-Cas9 Science 346 (6213), . [doi: /science ]
Gaj T., Gersbach C. A. and Barbas III C.F. (2013). ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol Vol. 31, No
Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, et al. (2016) Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922. PLoS ONE 11 (4): e doi: /journal.pone
Song G., Jia, M., Chen, K., Kong, X., Khattak, B., Xie, C., Li, A., and Mao, L. (2016). CRISPR/Cas9: A powerful tool for crop genome editing. The Crop Journal
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, j. A., and Charpentier, E. (2012).A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816