Barley as a model for cereal engineering and genome editing. Wendy Harwood

Transcription

1 Barley as a model for cereal engineering and genome editing Wendy Harwood MonoGram 29 th April 2015

2 BRACT Transformation Platform Over-expression of single genes RNAi based silencing Promoter testing Key crops: Barley Brassica oleracea Brassica napus Wheat Potato Brassica rapa Introduction of multiple genes Large scale cereal engineering Genome editing

3 Key requirements for an efficient transformation system: Highly regenerable target tissue amenable to Agrobacterium-mediated transformation. Simple and rapid transformation procedure. Minimum number of steps. Efficient selection system no escapes. High efficiency of transformation. Easy rooting and transfer to soil. High quality T0 transgenic plants No chimeric plants High proportion of single copy plants Efficient transmission of transgenes to the progeny, expected segregation and no loss of expression.

4 Genetic modification of barley Golden Promise 3 days co-cultivation 6 weeks callus induction 2 weeks transition 2-4 weeks regeneration Selection on 50mg/l hygromycin Average efficiency 40% Total weeks

5 Barley transformation pipeline 150 constructs + 80 rapid testing (19,000 immature embryos 2015) Escapes extremely rare Aim to take 15 independent transgenic plants / construct Select the homozygous T1 plants together with null controls if required for further analysis Germinate T1 seed, PCR to identify nulls, qpcr for copy number to identify homozygous plants Transfer to soil and determine transgene copy numbers initial expression analysis Grow to maturity and harvest T1 seed 7-8 months from transformation to harvest Seed will germinate as soon as it is fully dry. Seed can be germinated on hygromycin to identify positive plants.

6 Number of individuals, % Number of individuals Copy number of 249 T0 barley plants < HYG copy number < HYG copy number

7 Transformation efficiency % Effect of T-DNA size on transformation efficiency T-DNA size (Kb)

8 Number of independent transgenic plants Effect of T-DNA size on transformation efficiency T-DNA size

9 Summary so far: Highly efficient and straightforward Agrobacterium-mediated transformation in barley Large-scale cereal engineering projects requiring many constructs to be introduced are possible in barley T-DNA sizes up to 40Kb are OK

10 Genome editing or genome engineering technologies These allow: Targeted mutations Targeted insertions Gene replacements Technologies rely on sequence specific nucleases that generate double-stranded DNA breaks in the gene to be targeted. 1. Zinc-finger nucleases - allows plant researchers to target and replace specific genes. 2. TALENs (transcription activator like effector nucleases) have great promise as broadly applicable genome editing tools 3. CRISPRs (clustered regulatory interspaced short palindromic repeat) the latest technology with wide potential application Curtin et al., (2012) Genome Engineering of Crops with Designer Nucleases. The Plant Genome, 5: 42-50

11 CRISPR / Cas The most recent tool for genome editing is the CRISPR/Cas system (clustered regularly interspaced short palindromic repeats). Uses a guide RNA to direct the nuclease. Discovered as a bacterial defence system against invading foreign DNA. Cas targeting systems could be very significant in future gene therapy applications. They could also have huge implications for crop improvement. They have been shown to work in rice, wheat, maize and barley. Non-homologous end joining Sampson & Weiss (2013) Bioessays 36:34-38 Homology-directed repair Advantages over previous genome editing systems include easy to design, flexible, affordable and efficient.

12 Cas9 Cas9 nickases dcas9 dcas9 GFP Activator CRISPR / Cas9 applications Targeted mutagenesis Insertions Deletions Precise insertions or gene replacements In vivo imaging of specific genomic loci Up-regulation dcas9 Repressor Silencing (CRISPRi)

13 Targeted Mutagenesis in Barley using CRISPR/Cas9 Golden Gate cloning used to create constructs Level 1 components: Wheat U6 promoter Guide RNA including 20bp target sequence Maize Ubiquitin promoter Cas9 with C-terminal NLS T-nos 35S promoter HptII (Hygromycin Resistance) T-35S Combined to create a level 2 construct For the first barley target, two constructs were prepared each containing pairs of guide RNAs with one guide in common between the two constructs.

14 Single guide RNA strategy Screen to detect indels caused by imperfect NHEJ repair Paired guide RNA strategy PCR FORWARD RE TARGET 1 TARGET 2 Target Gene PCR REVERSE Screen to detect deletions corresponding to distance between two targets

15 Single guide T0 screen. Enriched for mutations by restriction digestion followed by PCR. Bands in + (restriction) lanes were cloned and sequenced to reveal indels at target locus. Paired guide T0 screen. Enrich for mutations by restriction digest followed by PCR. Band shift to lower position indicates deletion at target locus confirmed by sequencing 40 T0 lines screened at the early plantlet stage, 4 active lines found

16 Active lines analysed in T1 by PCR and direct sequencing (no enrichment) 3/4 T0 active lines showed presence of heterozygous/homozygous target site mutations in T1 plants. All correspond to frame shift or significant deletions. The best T0 line had 18 putative heterozygote mutants and 2 homozygotes from a total of 96 T1 plants screened Typical sequence profile of likely heterozygote in T1. Target sequence underlined in red. Note double peaks have similar height. Double peaks begin 4 bases before the PAM motif (the predicted cut point of Cas9)

17 Barley summary A diploid model cereal and an important crop Highly efficient and straightforward Agrobacterium-mediated transformation Large-scale cereal engineering projects possible T-DNA sizes up to 40Kb are OK CRISPR / Cas genome editing possible in barley

18 Acknowledgements Mark Smedley Alison Hinchliffe Tom Lawrenson Penny Hundleby Di Liu Asyraf Hatta Rob Blundell Eleni Soumpourou Andrey Korolev Maria-Luisa Amador-Delgado Oluwaseyi Shorinola Cristobal Uauy