Gene Editing in Cereals. Emma Wallington

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1 Gene Editing in Cereals Emma Wallington

2 NIAB Group 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

3 NIAB Crop Transformation Wheat, barley Oilseed rape Rice, potato Development/improvement of tissue culture & transformation systems

4 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 applications Mali et al., 2013, Science

5 Identification of a target for gene editing in rice Rice hebibaaoc mutant is unable to establish AM symbiosis 170 kb-deletion containing 26 genes including D14L Introduction of D14L in d14l mutant WT d14l mutant Line 1 Line 2 A : arbuscule(nutrient exchange) HP: hyphopodium V : vesicle (fungal nutrient storage structure) Gutjhar et al., 2015, Science

6 D14L CRISPR lines Construct strategy: Vector Miao et al., 2013 Cell Research, 23: ZmUbi promoter, Cas9 : Codon optimized for rice, U3 promoter, HygR cassette Single guide RNA, designed with CRISPR-P program (

7 D14L CRISPR lines Mutation 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 * *

8 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

9 D14L CRISPR lines (T1generation) 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. -A/+T biallelic -TG homozygous +T homozygous Lesley Plucker

10 D14L complementation Re-transformation with Rice D14L promoter: D14L CDS: D14L terminator constructs restores AM colonization Lesley Plucker

11 NIAB Wheat Transformation Very efficient Agrobacterium transformation of wheat pipeline Embryo preparation and DNA delivery Throughput of>3000 independent transformed wheat plants per year Callus production 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 Plant regeneration Seed production Small rooted plantlets: 12 weeks T1 seed: months

12 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

13 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

14 Inheritance Ten T0 lines carried forward (3 edited, 7 nonedited) 293 T1 plants analysed No additional edits Segregation of edit in T1 plants T0 line Homozygous Heterozygous χ2 P WT edit edit value GE GE GE Two T1 lines selected T2 generation: T-DNA free lines identified T2 homozygous edited clean lines identified in 36 weeks T1 homozygous edit

15 Can Cas9 generate more edits? T2 homozygous edited embryos Callus induction & regeneration 251 plantlets

16 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

17 Somatic/Chimeric editing observed in barley 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

18 GE for candidate gene validation 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 pmm12 T-DNA 13Kb Sc4 promoter NptII Term OsU3-sg1 TaU3-sg2 TaU6-sg3 OsU6-sg4 Constitutive promoter Cas9 Term Selection cassette 4-guide stack Cas9 cassette

19 Ta putative male fertility gene CRISPR OsU3 grna1 * TaU6 grna3 TaU3 grna2* OsU6 grna4 * Anticipated deletions TaA/B/D: bp CAPS assay *

20 T0 plant analysis: >100 plants to screen Key: 1st PCR screen Clone PCR products & sequence WT fragment size, 1.6Kb smaller fragment sizes heterozygous (mutation/wt) biallelic mutation homozygous mutation Plant number Ta A Ta B Ta D con1 con2 con3 Ta A (bp) Ta B (bp) Ta D (bp) & -933

21 Ta A genome Ta B genome Ta D genome

22 plant Ta A Ta B Ta D 3-1,-1, , -4, (Hom) 5* +1 (Hom) -1 (Hom) -17, -62, WT, +118 missense, -946 WT 17* -3, -5-5, , -933 * Sterile phenotype

23 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 Pollen: WT Carpels: WT 5 3 WT WT

24 Wheat Blast

25 Wheat blast may affect barley, maize and several weeds but probably not rice (USA) Wheatblast pandemic timeline

26

27 Deploy gene editing in wheat to KO candidate S-genes Wheat blast infection assessment Capacity building and knowledge transfer

28 Crop specific strategies: Constructs nuclease & guides validated in the species of interest Germplasm- to avoid lengthy backcrossing

29 Crop specific strategies: mutation detection 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 Low cost, can be high throughput Labour intensive and /or expensive

30 Conclusions 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

31 NIAB Crop Transformation: Rhian Howells Matthew Milner Charline Soraru Melanie Craze Sarah Bowden Ruth Bates Uta Paszkowzki Jeongmin Choi Will Summers Lesley Plucker Anthony Keeling Nick Talbot Sophien Kamoun Thorsten Langner