CRISPR/Cas approach in modern plant breeding

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CRISPR/Cas9 approach in modern plant breeding:

CRISPR/Cas9 approach in modern plant breeding Mehmet Emin ÇALIŞKAN 1 , Sarbesh Das DANGOL 1 1 Department of Agricultural Genetic Engineering, Faculty of Agricultural Science and Technologies, Nigde University, 51240 Merkez, Nigde, Turkey June 15, 2016.

History of CRISPR/Cas technology:

History of CRISPR/ Cas technology CRISPR/Cas9 approach in modern plant breeding First identified in E. coli genome Biological function unraveled Confirmation of involvement in bacterial immunity From crRNA -tracrRNA to gRNA CRISPR/Cas9 technology in plants 1987 2005 Plant Genome Editing History 2012 2013 2007

Introduction:

Introduction CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9): Novel breakthrough Mutation in target: altering function/ interrogation RNA-guided nucleases utilized with adaptable specificities for endogenous genes CRISPR/Cas9 approach in modern plant breeding

TALEN vs ZFN and Gene Knockout:

TALEN vs ZFN and Gene Knockout CRISPR/Cas9 approach in modern plant breeding

CRISPR/Cas9: From bacteria to gene engineering tool:

CRISPR/Cas9: From bacteria to gene engineering tool CRISPR/Cas9 approach in modern plant breeding

The basic strategy of Cas9/sgRNA system :

The basic strategy of Cas9/ sgRNA system (Ding et al ., 2016) CRISPR/Cas9 approach in modern plant breeding

Importance of “seed sequence” of sgRNA:

Importance of “seed sequence” of sgRNA ( Bortesi and Fischer, 2015) CRISPR/Cas9 approach in modern plant breeding

Steps of generating CRISPR/Cas9 mutagenised plant line :

Steps of generating CRISPR/Cas9 mutagenised plant line CRISPR/Cas9 approach in modern plant breeding

Available online resources for CRISPR/Cas system:

Available online resources for CRISPR/ Cas system CRISPR/Cas9 approach in modern plant breeding

Steps of generating CRISPR/Cas9 mutagenised plant line :

Steps of generating CRISPR/Cas9 mutagenised plant line CRISPR/Cas9 approach in modern plant breeding

Steps of generating CRISPR/Cas9 mutagenised plant line :

Steps of generating CRISPR/Cas9 mutagenised plant line CRISPR/Cas9 approach in modern plant breeding

Genome editing vector:

Genome editing vector CRISPR/Cas9 approach in modern plant breeding pRGEB31  (Plasmid #51295) Addgene

Multiplex genome editing in rice:

Multiplex genome editing in rice CRISPR/Cas9 approach in modern plant breeding ( Lian et al .,2016)

Some CRISPR/Cas approaches:

Some CRISPR/ Cas approaches MLO (Mildew-resistance locus) genes targeted in wheat: resistance against powder mildew disease. (Wang et al ., 2014) USDA will not regulate a gene edited mushroom that resists browning. Target: polyphenol oxidase ( PPO ). (Nature News, April 2016) German authorities confirmed GE canola as non-GMO. (Reviewed in Huang et al ., 2016) CRISPR/Cas9 approach in modern plant breeding

Some CRISPR/Cas approaches:

Some CRISPR/ Cas approaches Cucumis sativus L.: elF4E gene knocked out conferred resistance against Zucchini yellow mosaic virus  and  Papaya ring spot mosaic virus. ( Chandrasekaran et al . 2016 ) Glycine max : ALS ( acetolactate synthase ) mutation via HDR insertion conferred Chlorosulfuron resistance. (Li et al ., 2015) Rice Acetolactate synthase ( ALS ) gene targeted at two sites using HDR. Homozygous herbicide-resistant plants in one generation. (Sun et al ., 2016) CRISPR/Cas9 approach in modern plant breeding

History of CRISPR arrays:

History of CRISPR arrays CRISPR/Cas9 approach in modern plant breeding

Cas9 Nickase:

Cas9 Nickase Mutation ( A sp Ala) Creates nick instead of DSB at target site . CRISPR/Cas9 approach in modern plant breeding

Inactive dCas9:

Inactive dCas9 For RNA guided transcription regulation instead of genome editing. (CRISPR interference) CRISPRi and CRISPR activation ( CRISPRa ) CRISPR/Cas9 approach in modern plant breeding

Development of an active FokI-dCas9 fusion architecture:

Development of an active FokI-dCas9 fusion architecture CRISPR/Cas9 approach in modern plant breeding

Truncated Guide RNAs (truRNA) and GG+20nt gRNA:

Truncated Guide RNAs ( truRNA ) and GG+20nt gRNA sgRNA variants with 17-nt shorter target complementarity . Decreased undesired mutagenesis by 5,000-fold (Fu et al., 2014). CRISPR/Cas9 approach in modern plant breeding

Ribozyme-gRNA-Ribozyme (RGR):

Ribozyme-gRNA-Ribozyme (RGR) Self-catalyzed cleavage; tissue-specific genome editing possible. CRISPR/Cas9 approach in modern plant breeding

Polycistronic tRNA-gRNA (PTG/Cas9):

Polycistronic tRNA-gRNA (PTG/Cas9) Array of tandem tRNA-gRNA units. Target multiple sites. CRISPR/Cas9 approach in modern plant breeding

Cis gene stacking:

Cis gene stacking Sequential addition of transgenes at the same locus. All transgenes inherited as a single locus. CRISPR/Cas9 approach in modern plant breeding

Transgene-free homozygous GE plants in just one generation:

Transgene -free homozygous GE plants in just one generation CRISPR/Cas9 approach in modern plant breeding ( Khatodia et al ., 2016)

Conclusion:

Conclusion CRISPR/Cas9 approach in modern plant breeding CRISPR/Cas9 method modifies genes rapidly, easily and efficiently. Gene edited crops needless to be regulated. Mutagenesis , gene regulation , gene stacking.   Homozygous, stable and heritable. High yielding combating biotic/abiotic stresses.

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