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.

1. Precision Plant Breeding using Genome Editing Technologies
Caixia Gao
June 28, 2018

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. 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. Precise genome modifications are achieved by harnessing DNA
double strand break repair pathways
Shan et al., Nature Protoc, 2014

5. Wheat is recalcitrant to conventional genetic manipulation
17.1Gb 2.3Gb 1.2Gb 0.45Gb
Gil-Humanes et al., Nature Biotechnol, 2014

6. Powdery mildew is one of the most destructive diseases in wheat

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. Engineered TALENs to target three TaMLO homoeoalleles
Ubi-1 TALEN-L TALEN-R NOST2AT-MLO
Wang et al., Nature Biotechnol, 2014

9. The tamlo homozygous mutants obtained by self-pollinations
Wang et al., Nature Biotechnol, 2014

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. 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. Editing rice flavor
Shan et al., Plant Biotech J. 2015

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. 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. Targeted mutagenesis using CRISPR/Cas9 RNPs in wheat
Liang et al., Nature Comm., 2017

16. Base editing mediated guide RNA-programmed C to T conversion
Komor et al., Nature, 2016
• High efficiency
• No DSBs
• No donor DNA

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. Targeted TaLOX2 gene in wheat
Zong et al., Nature Biotechnol, 2017

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. 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. • 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. 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. 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. Acknowledgements
Funding
Collaborators
Jin-Long Qiu, Inst of Microbiology, CAS
Jiayang Li, IGDB, CAS
Daowen Wang, IGDB, CAS
Dan Voytas, Minnesota University