gene editing in cereals - csiro research · genome editing with crispr cas9 • using existing...
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• NIAB established in 1919 by charitable donations for ‘the improvement of crops .. with higher….. genetic quality’
• A charitable company limited by guarantee
• We provide independent science-based research and information for the agriculture and horticulture sectors
• Integration of TAG, CUF, EMR
• NIAB Innovation Farm, NIAB International
NIAB Group
NIAB Crop Transformation• Wheat, barley
•Oilseed rape
• Rice, potato
• Development/improvement of tissue
culture & transformation systems
Genome editing with CRISPR Cas9
• Using existing tools for gene characterisation in rice
• Validate tools for implementation with our high
throughput stable wheat transformation pipeline
• Single homoeologue KO
• KO all 3 homoeologues
• Development of marker free lines
• Use in practical applicationsMali et al., 2013, Science
Identification of a target for gene editing in rice
Gutjhar et al., 2015, Science
• Rice hebibaAOC mutant is unable to establish AM symbiosis
• 170 kb-deletion containing 26 genes including D14L
WT
d14l mutant
Introduction of D14L in d14l mutant
Line 1 Line 2
A : arbuscule (nutrient exchange)
HP: hyphopodium
V : vesicle (fungal nutrient storage structure)
D14L CRISPR lines
• Construct strategy:
• Vector Miao et al., 2013 Cell Research, 23: 1233-1236
• ZmUbi promoter, Cas9 : Codon optimized for rice, U3 promoter, HygR cassette
• Single guide RNA, designed with CRISPR-P program
(http://crispr.hzau.edu.cn/CRISPR2/)
D14L CRISPR linesMutation identification
T0 generation
• Sequencing of the predicted edit site around PAM sequence
• ~90% of the plants have mutations
• Homozygous mutation identified, resulting in frame shift *
*
D14L CRISPR lines
*Black shading indicates the sequence is identical to wild type D14L
*To assess the consequence of the transcript, quantitative RT-PCR analysis is in progress.Jeongmin Choi
D14L CRISPR lines (T1generation)
-TG homozygous-A/+T biallelic +T homozygous
Lesley Plucker
T1 generation
• PCR to select plants which do not contain T-DNA
• Sequencing of the edit site around PAM sequence to identify homozygous lines
• D14L CRISPR lines were unable to become colonized, confirming the role of D14L in the
initiation of fungal infection.
D14L complementation
Re-transformation with Rice D14L promoter: D14L CDS: D14L terminator
constructs restores AM colonizationLesley Plucker
Embryo
preparation
and DNA
delivery
Callus
production
Plant
regeneration
Seed
production
NIAB Wheat Transformation
• Very efficient Agrobacterium transformation of wheat
pipeline
• Throughput of >3000 independent transformed wheat
plants per year
• Academic research and CTS
• Continuous development of germplasm and technical
resources
• widen the pool of germplasm
• promoter characterisation
• implementation of new technology e.g. gene editing
Small
rooted
plantlets:
12 weeks
T1 seed:
6.5 - 7
months
Wheat gene editing• New suite of constructs designed and tested with multiple guides
• Constructs fit with our standard Agrobacterium–mediated transformation system
• Single guides used to focus on off target effects and stability
• Single target gene – PDS
– 95-96% identity between the 3 homoeologues
– Test wheat and barley with same construct
Wheat editing results – single guides
• 6 constructs generated 283 T0 plants, with 34 plants edited
• PCR products cloned and Sanger sequenced
• 5-18 % efficiency
• 50% of edits are 1bp indels
• 14% are biallelic
• Largest edit is 34bp insertion
• No homozygous edits
• No off-target effects
• All genomes edited by a single guide, but not simultaneously
• Ten T0 lines carried forward (3 edited, 7 non-
edited)
• 293 T1 plants analysed
• No additional edits
• Two T1 lines selected
• T2 generation: T-DNA free lines identified
• T2 homozygous edited clean lines identified
in 36 weeks
T1 homozygous edit
Inheritance
T0 line Homozygous
edit
Heterozygous
editWT
χ2 P
value
GE1-2 8 8 3 0.212
GE1-31 6 14 8 0.687
GE7-5 6 14 9 0.721
Segregation of edit in T1 plants
Can Cas9 generate more edits ?
T2 homozygous
edited embryos
Callus induction & regeneration
251 plantlets
Barley gene editing
• HvPDS editing with a wheat construct
• 15% of plants edited, compared with 5% in wheat
• Edits produced are much larger with up to 350bp deleted
from the single gRNA
• Plants show more than 2 different genotypes, suggesting
high levels of chimerism
• Chimeric bleaching observed
• Chimeric photobleaching in edited plants
• Chimeric photobleaching develops in plants with no edit
• DNA extracted from white, green and striped leaf material
• White tissue exhibits homozygous editing
• Striped tissue is heterozygous
• Green can be either wild type or heterozygous
• DNA resampled from all lines
• Plants previously assigned as edited are now wild type
• Plants previously wild type are now editing
Somatic/Chimeric editing observed in barley
• Candidate genes (putative male-fertility genes) identified from stamen
specific RNAseq library
• Will KO of candidate genes result in male-sterility?
• Candidate gene 1: 17kb, 33 exons
• 97% identity in the exonic regions
• 4 guides designed to target exons 3 & 5 across all 3 homoeologues
GE for candidate gene validation
pMM12 T-DNA
13Kb
Sc4
promoter NptII Term
Constitutive
promoter Cas9 TermOsU3-sg1 TaU3-sg2 TaU6-sg3 OsU6-sg4
Selection cassette 4-guide stack Cas9 cassette
• Anticipated deletionsTaA/B/D: 44-1036bp
• CAPS assay *
TaU6 gRNA3
OsU3 gRNA1 *
TaU3 gRNA2*
OsU6 gRNA4 *
1000 2000 3000 4000 5000
Ta putative male fertility gene CRISPR
T0 plant analysis:
• >100 plants to screen
Key:1st PCR screen
WT fragment size, 1.6Kb
smaller fragment sizes
Clone PCR
products &
sequence
heterozygous
(mutation/WT)
biallelic mutation
homozygous mutation
Ta A
(bp)
Ta B
(bp) Ta D (bp)
-940
-940
-966 -940
-1029 -938
-1067
-964
+118 -946
-940 -939 & -933
Plant
number Ta A Ta B Ta D
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
con1
con2
con3
Ta A genome
Ta B genome
Ta D genome
plant Ta A Ta B Ta D
3 -1 , -1, -179 -3, -4, -966 -940 (Hom)
5 * +1 (Hom) -1 (Hom) -17, -62, +1
15 WT, +118 missense, -946 WT
17 * -3, -5 -5, -1 -939, -933
* Sterile phenotype
• Phenotype: plants 5 and 17 were male-sterile; the
other 16 plants were fertile
• Aberrant pollen morphology observed
• Otherwise plants developmentally normal and able
to receive viable pollen from donor plants
5
WT
WT
5
Carpels:
Pollen:
WT3 WT 17 17
Wheat Blast
Wheat blast may
affect barley, maize
and several weeds
but probably not rice
(USA)
Wheatblast pandemic timeline
• Deploy gene editing in wheat to KO candidate S-genes
• Wheat blast infection assessment
• Capacity building and knowledge transfer
• Constructs – nuclease & guides validated in the species of interest
• Germplasm - to avoid lengthy backcrossing
Crop specific strategies:
• Initial screening
– Target amplicon size changes, CAPS, T7 assay
– Phenotyping
• Rice & barley (diploid)
– Direct sequencing of PCR products
– Sequence cloned PCR products
• Wheat (hexaploid)
– Homoeologue specific PCR required, not always easy to design
– Requires specificity confirmation using NT DNA (CS)
– CAPS assay, not always possible to design
– New sequencing strategies and technologies
Crop specific strategies: mutation detection
Low cost, can be
high throughput
Labour intensive
and /or expensive
Rice
• Efficient, T0 homozygous material
• Phenotypes observed in T0
• Marker free lines identified
Wheat
• Efficient, T0 homozygous material
• Marker free lines identified
• KO of all 3 homeologues achieved
• Phenotypes observed in T0
Barley
• Mutations identified, but concerns regarding stability and somatic editing
Conclusions
NIAB Crop Transformation:Rhian Howells Melanie Craze
Matthew Milner Sarah Bowden
Charline Soraru Ruth Bates
Uta Paszkowzki
Jeongmin Choi
Will Summers
Lesley Plucker
Nick Talbot
Sophien Kamoun
Thorsten Langner
Anthony Keeling