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

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

4. 1234567891011121314151617MPN
PCR Screening transgenic plants for Cas9 integration
PCRamplificationoftargetregionfromtheCas9positivetransgeneiclines
1 2 3 4 5 6 7 10 11 13 14 15168 9 12 17
Generating Cas9 and sgRNA expressing transgenic rice

5. Single allelic rice TB1 (Os03g49880) locus editing in different MTU1010 edited lines
GGCGGGCAAGGCGTCGACGCCGG

7. GCAAGAACAATTGAGGCACCTTGG
ControlriceplantEditedriceplant Bi-allelic DEP1 (Os09g26999) editing in rice MTU1010 cultivar

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

11. Bi-allelic rice TB1 (Os03g49880) locus editing in different MTU1010 edited lines

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

15. GACGTCGAGCTCTAGCACGTTGG
TWO NUCLETIDE DELEATION IN CKX2 GENE IN RICE MTU1010

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

21. OsDEP1 (Os09g26999)
knockout rice line
OsTB1 (Os03g49880)
knockout rice line

22. RBLB
WDV ALS repair template
A P W S
96 171 548 627
122 197 574 653
Four known ALS herbicide resistance endowing mutations
Chlorsulfuron
Bensulfuron
methyl
Imazaquin
Imazapyr
Bispyribac
sodium
Pyrithiobac
sodium
Pyriminobac
A/V P/S W/L S/I
10
190
55
14
20
1
140
300
9100
25
3.7
5.3
56
13
8300
9100
56
14
2800
2900
6500
4.2
43
21
14
83
350
3300
2.4
73
6.8
10
41
200
2500
200
14000
45
10
16000
9100
13000
Arabidopsis
Rice
Viral based vectors for delivery of repair template
Rep expression cassette
Rep terPromoter
sgRNA

23. Introduction of of precise SNP (C to T, or G to A) mutations
without any double strand break and without HDR template

24. THANK YOU