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

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

CRISPR Cas9

Cas9 protein Doudna and Charpentier (2014)

Endogenous cellular DNA Repair machinery

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

-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

How to use it for genome editing Vector construction

Adavantages Applications

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

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

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.

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

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

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

Analysis of the agronomic traits of 6 homozygous T2 mutant lines

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.

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: 10.1126/science.1258096] 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. 7 398 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): e0154027. doi:10.1371/journal.pone.0154027 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 75-82. 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