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Precision Plant Breeding using Genome Editing Technologies
Caixia Gao
June 28, 2018
Genome editing techniques provide new opportunities for crop breeding
Conventional breeding Mutation breeding Transgenesis...
Sequence-specific nucleases enable efficient genome engineering
ZFN
TALEN
CRISPR/Cas9
• Zinc finger protein (DNA binding d...
Precise genome modifications are achieved by harnessing DNA
double strand break repair pathways
Shan et al., Nature Protoc...
Wheat is recalcitrant to conventional genetic manipulation
17.1Gb 2.3Gb 1.2Gb 0.45Gb
Gil-Humanes et al., Nature Biotechnol...
Powdery mildew is one of the most destructive diseases in wheat
Choosing to target MLO loci in bread wheat
 Editing MLO genes in wheat may provide the opportunity to breed
varieties wit...
Engineered TALENs to target three TaMLO homoeoalleles
Ubi-1 TALEN-L TALEN-R NOST2AT-MLO
Wang et al., Nature Biotechnol, 20...
The tamlo homozygous mutants obtained by self-pollinations
Wang et al., Nature Biotechnol, 2014
Loss of TaMLO function confers wheat resistance to powdery mildew
WT tamlo-aabbdd
Detached leaves
Wheat plant
Wang et al.,...
Accelerating corn breeding
 Male fertility gene knock out in corn
 Necessary for new hybrid seed production systems
Li e...
Editing rice flavor
Shan et al., Plant Biotech J. 2015
Conventional genome editing in plants
 No foreign DNA remains in mutant plant after segregation away of
the nuclease tran...
DNA-free genome editing in wheat
 Wheat immature embryos were treated with purified Cas9 protein or in vitro transcript
C...
Targeted mutagenesis using CRISPR/Cas9 RNPs in wheat
Liang et al., Nature Comm., 2017
Base editing mediated guide RNA-programmed C to T conversion
Komor et al., Nature, 2016
• High efficiency
• No DSBs
• No d...
Targeting OsCDC48 gene in rice
 C –T conversion efficiency 40/92=43.5%
 The deamination window 3 to 8
 No indels were o...
Targeted TaLOX2 gene in wheat
Zong et al., Nature Biotechnol, 2017
Gaudelli et al., Nature, 2017
• High purity; No DSBs; No donor DNA
• High efficiency in human cells
Scope and overview of ...
Plant ABE edited rice plants conferred herbicide resistance
 OsACC-T1 with T7>C7
converts C2186 to
R2186.
 Heterozygous ...
• New DNA gene
expression cassette
(foreign DNA)
• Final product does
contain foreign DNA
• New DNA gene
expression casset...
Conclusions
 Genome editing can effectively induce targeted mutations in plant
genomes
• Precise location
• Many alleles ...
Future perspectives
Economic, regulatory and societal benefits
 Reduced costs for precise and efficient molecular breedin...
Acknowledgements
Funding
Collaborators
Jin-Long Qiu, Inst of Microbiology, CAS
Jiayang Li, IGDB, CAS
Daowen Wang, IGDB, CA...
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An overview of agricultural applications of genome editing: Crop plants

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The presentation gives an overview of genome editing applications in relation to crop plants. The aim is to have a better understanding of the specific features of genome editing in comparison with classical breeding and genetic engineering techniques. It will give an overview of some examples of agricultural applications that may be on or close to the market or under research and development. It will also consider the possibility of foreseeing future applications (e.g. variations in CRISPR/Cas applications, DNA-free application, agricultural pest control), if possible.

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An overview of agricultural applications of genome editing: Crop plants

  1. 1. Precision Plant Breeding using Genome Editing Technologies Caixia Gao June 28, 2018
  2. 2. Genome editing techniques provide new opportunities for crop breeding Conventional breeding Mutation breeding Transgenesis Genome editing narrowing genetic diversity time consuming; expensive screens regulatory complexity; high cost precise, predictable variations Modified from Podevin et al, EMBO Rep, 2012
  3. 3. Sequence-specific nucleases enable efficient genome engineering ZFN TALEN CRISPR/Cas9 • Zinc finger protein (DNA binding domain) fused with a catalytic nuclease domain • 1st engineered endonucleases used to edit genes • Cas9 is the nuclease protein that cuts the DNA • The site specificity comes from the guide RNA, which can be designed and synthesized easily • TAL effector (DNA binding domain) fused with a catalytic nuclease domain • easier to engineer than ZFNs Voytas and Gao, PLoS Biol, 2014
  4. 4. Precise genome modifications are achieved by harnessing DNA double strand break repair pathways Shan et al., Nature Protoc, 2014
  5. 5. Wheat is recalcitrant to conventional genetic manipulation 17.1Gb 2.3Gb 1.2Gb 0.45Gb Gil-Humanes et al., Nature Biotechnol, 2014
  6. 6. Powdery mildew is one of the most destructive diseases in wheat
  7. 7. Choosing to target MLO loci in bread wheat  Editing MLO genes in wheat may provide the opportunity to breed varieties with broad-spectrum and durable resistance to Bgt Barley Arabidopsis Tomato mlo mutants resistance to powdery mildew
  8. 8. Engineered TALENs to target three TaMLO homoeoalleles Ubi-1 TALEN-L TALEN-R NOST2AT-MLO Wang et al., Nature Biotechnol, 2014
  9. 9. The tamlo homozygous mutants obtained by self-pollinations Wang et al., Nature Biotechnol, 2014
  10. 10. Loss of TaMLO function confers wheat resistance to powdery mildew WT tamlo-aabbdd Detached leaves Wheat plant Wang et al., Nature Biotechnol, 2014
  11. 11. Accelerating corn breeding  Male fertility gene knock out in corn  Necessary for new hybrid seed production systems Li et al., J Gemonics Genet, 2016
  12. 12. Editing rice flavor Shan et al., Plant Biotech J. 2015
  13. 13. Conventional genome editing in plants  No foreign DNA remains in mutant plant after segregation away of the nuclease transgene mlo mlo mlo CRISPR CRISPR transgenic knock-out plant CRISPR Delivery mlo mlo mlo Transgene-free mutant Selfing/Crossing Genome A 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 MLO MLO MLO Genome B Genome D Wild type wheat plant Disadvantages:  Potential off-target effects  Time-consuming for segregation  Impossible for vegetatively propagated plants  Small DNA insertion Zhang et al., Nature Comm., 2016
  14. 14. DNA-free genome editing in wheat  Wheat immature embryos were treated with purified Cas9 protein or in vitro transcript Cas9 and gRNA  Reduced off-target effects; no exogenous DNA used Zhang et al., Nature Comm., 2016; Liang et al., Nature Comm., 2017
  15. 15. Targeted mutagenesis using CRISPR/Cas9 RNPs in wheat Liang et al., Nature Comm., 2017
  16. 16. Base editing mediated guide RNA-programmed C to T conversion Komor et al., Nature, 2016 • High efficiency • No DSBs • No donor DNA
  17. 17. Targeting OsCDC48 gene in rice  C –T conversion efficiency 40/92=43.5%  The deamination window 3 to 8  No indels were observed in the target region Zong et al., Nature Biotechnol, 2017
  18. 18. Targeted TaLOX2 gene in wheat Zong et al., Nature Biotechnol, 2017
  19. 19. Gaudelli et al., Nature, 2017 • High purity; No DSBs; No donor DNA • High efficiency in human cells Scope and overview of base editing by an A•T to G•C base editor
  20. 20. Plant ABE edited rice plants conferred herbicide resistance  OsACC-T1 with T7>C7 converts C2186 to R2186.  Heterozygous mutant was conferred resistance to herbicide. Li et al. Genome Biology 2018
  21. 21. • New DNA gene expression cassette (foreign DNA) • Final product does contain foreign DNA • New DNA gene expression cassette (native or foreign) • Final product may contain foreign DNA • Small change to native DNA • Final product no foreign DNA Product similar to conventional breeding SDN-1 SDN-2 SDN-3 GMO • Small change to native DNA • Final product no foreign DNA Product similar to conventional breeding Product precisely modified compared to GM Product genetically modified • Small and large changes to native DNA • Final product no foreign DNA Conventional Genetically ModifiedGenome Editing Product produced by conventional breeding Crossing Not regulated or regulated?Not regulated Regulated Overview of genome-edited products
  22. 22. Conclusions  Genome editing can effectively induce targeted mutations in plant genomes • Precise location • Many alleles at the same time, also in polyploid crops • Reduce non-specific off-target cleavage • Perform highly efficient and site-specific C to T base editing in plants  Constructs can be segregated away by crossing; no random insertion of Cas9 or gRNA into plant genome by using RNP or IVTs for knockout and base editing  Final products • Identical to the mutants obtained by ‘conventional’ mutagenesis
  23. 23. Future perspectives Economic, regulatory and societal benefits  Reduced costs for precise and efficient molecular breeding  Eliminate or significantly reduce regulatory requirements • Regulate products of NBT consistently with products from conventional breeding, if they are indistinguishable • Regulation for safe use focuses on characteristics of the plant and phenotype and intended use  Alleviate public concerns about gene edited crops
  24. 24. Acknowledgements Funding Collaborators Jin-Long Qiu, Inst of Microbiology, CAS Jiayang Li, IGDB, CAS Daowen Wang, IGDB, CAS Dan Voytas, Minnesota University

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