Editing rice-genome with CRISPR/Cas9: To improve agronomic traits for increased crop productivity by MK Reddy during the Regional Expert Consultation on Gene Editing in Agriculture and its Regulations Technical Session III
YHR Fall 2023 Issue (Joseph Manning Interview) (2).pdf
Editing rice-genome with CRISPR/Cas9: To improve agronomic traits for increased crop productivity
1. M K REDDY
GROUP LEADER
CROP IMPROVEMENT GROUP
INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY
Editing rice-genome with CRISPR/Cas9:
To improve agronomic traits for increased crop productivity
2. One can edit any genome by expressing Cas9 Protein and gRNA
Maize
Ubiquitin
Promoter
Cas9
NOS
terminator
Rice U6
Promoter sgRNA Terminator
3. MTU1010 high yielding popular rice mega-verity
Cas9 & sgRNA encoding genes
Together cloned into single
plant transformation vector
Agrobacterium mediated
Rice transformation & regeneration
Cas9 & sgRNA expressing
Transgenic rice plants were identified
Targeted genome edited
rice plants were identified
Transgenic component free
genome edited rice lines generated
Through sequence analysis
Through Molecular analysis
Segregating out the
transgenic component
Cas9 and sgRNA
Through crossing, selfing
and subsequent selection
Putative transgenic rice plants generated
OsDEP1 (Os09g26999)
knockout rice line
OsTB1 (Os03g49880)
knockout rice line
OsCKX2 (Os08g0509600)
knockout rice line
OsGW2 (Os02g2914720)
knockout rice line
Non-transgenic high yielding genome edited rice lines
Background work done related to the project
9. MTU1010 high yielding popular rice mega-verity
Cas9 & sgRNA encoding genes
Together cloned into single
plant transformation vector
Agrobacterium mediated
Rice transformation & regeneration
Cas9 & sgRNA expressing
Transgenic rice plants were identified
Targeted genome edited
rice plants were identified
Transgenic component free
genome edited rice lines generated
Through sequence analysis
Through Molecular analysis
Segregating out the
transgenic component
Cas9 and sgRNA
Through crossing, selfing
and subsequent selection
Putative transgenic rice plants generated
OsDEP1 (Os09g26999)
knockout rice line
OsTB1 (Os03g49880)
knockout rice line
OsCKX2 (Os08g0509600)
knockout rice line
OsGW2 (Os02g2914720)
knockout rice line
Non-transgenic high yielding genome edited rice lines
Background work done related to the project
10.
11. Bi-allelic rice TB1 (Os03g49880) locus editing in different MTU1010 edited lines
12.
13. MTU1010 high yielding popular rice mega-verity
Cas9 & sgRNA encoding genes
Together cloned into single
plant transformation vector
Agrobacterium mediated
Rice transformation & regeneration
Cas9 & sgRNA expressing
Transgenic rice plants were identified
Targeted genome edited
rice plants were identified
Transgenic component free
genome edited rice lines generated
Through sequence analysis
Through Molecular analysis
Segregating out the
transgenic component
Cas9 and sgRNA
Through crossing, selfing
and subsequent selection
Putative transgenic rice plants generated
OsDEP1 (Os09g26999)
knockout rice line
OsTB1 (Os03g49880)
knockout rice line
OsCKX2 (Os08g0509600)
knockout rice line
OsGW2 (Os02g2914720)
knockout rice line
Non-transgenic high yielding genome edited rice lines
Background work done related to the project
14. Cytokinin Oxidase Regulates Rice spike architecture and increase he grain number
• The rice OsCKX2 differentially expresses in rice inflorescence
• Cytokinin oxidase degrades cytokine and negatively regulates the inflorescence branching
• Absence of OsCKX2 expression cytokine accumulation and increases the grain number
Ashikari et al 2005 Science: 309, 741-745
17. MTU1010 high yielding popular rice mega-verity
Cas9 & sgRNA encoding genes
Together cloned into single
plant transformation vector
Agrobacterium mediated
Rice transformation & regeneration
Cas9 & sgRNA expressing
Transgenic rice plants were identified
Targeted genome edited
rice plants were identified
Transgenic component free
genome edited rice lines generated
Through sequence analysis
Through Molecular analysis
Segregating out the
transgenic component
Cas9 and sgRNA
Through crossing, selfing
and subsequent selection
Putative transgenic rice plants generated
OsDEP1 (Os09g26999)
knockout rice line
OsTB1 (Os03g49880)
knockout rice line
OsCKX2 (Os08g0509600)
knockout rice line
OsGW2 (Os02g2914720)
knockout rice line
Non-transgenic high yielding genome edited rice lines
Background work done related to the project
18. GW2 QTL for rice grain and weight encodes a RING-type E3 ubiquitin ligase
GW2 loss of function increased cell divisions in spikelet hull resulting in enhanced grain width, weight and yield
GW2: Os02g14720
19. PCR fragment cut with Sca1 (AGTACT) edited line will cut control won’t Cut