Gene Editing in Cereals

Emma Wallington

emma.wallington@niab.com

• 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