New Plant Breeding Techniques Group 1 Targeted Mutagenesis

Transcription

1 WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN New Plant Breeding Techniques Group 1 Targeted Mutagenesis Maria Lusser Joint Research Centre, European Commission Workshop Comparative situation of new plant breeding techniques September 2011, Seville, Spain 1

2 Techniques WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 2 Zinc Finger Nuclease technology Oligonucleotide directed mutagenesis (ODM) Meganuclease technique TALE technique (Transcription activator like effector proteins)

3 Spontaneous mutations & mutagenesis through ionizing radiation or chemical mutagens WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 3 Spontaneous mutations Induction by treatment of plant parts (seeds, pollen, vegetative parts e.g. buds) - with ionizing radiation (X-rays or gamma rays) - or chemical mutagens (sodium azide, ethyl methyl sulfonate)

4 Spontaneous mutations & mutagenesis through ionizing radiation or chemical mutagens WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 4 Consequences can be - chromosome breakage, translocation and elimination prevalent with irradiation - chromosome (segment) doubling - chromosome - single base-pair changes (substitution, insertion prevalent with chemical and deletion) mutagenes Further propagation or backcrossing and selection necessary Van de Wiel, C. et al., Traditional plant breeding techniques, Wageningen UR Plant Breeding, Report 338 (2010)

5 ZFN-1 technique WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 5 Zinc finger nucleases are chimeric proteins Zinc finger domain (recognising specific DNA sequence) & nuclease cutting double-stranded DNA ZFN-1 technology: - delivery of genes encoding ZFNs (without repair template) - site-specific double-strand break (DSB) - natural repair process (through non-homologous end-joining NHEJ) - mutation in one or a few bp, short deletions or insertions (site-specific but random change)

6 ZFN-2 technology WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 6 ZFN-2 technology: - delivery of genes encoding ZFNs & repair template (DNA stretch of a few kbp) - site-specific double-strand break (DSB) - natural repair process (through homomolgous recombination HR) - mutation in one or a few bp, short deletions or insertions (site-specific and specific change)

7 ZFN-1 technique WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 7 ZFN - DNA cutting domain ZFN - DNA binding domain Double strand break (DSB) Repair by NHEJ (error prone repair) Source: e.g.: Point mutation

8 ZFN-2 technique WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 8 Zinc Finger Nuclease (ZFN) Double strand break (DSB) Repair template with one bp mismatch Repair by homologous recombination (HR) Source: e.g.: Point mutation

9 ZFN-2 WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 9 Delivery of repair template - as DNA Gene insertion in the case of delivery as DNA vector construct encoding ZFN? DSB Delivery of ZFN: - DNA vector construct encoding ZFN - RNA encoding ZFN - as protein Repair by NHEJ Transgenic intermediate plant? Regeneration/ Selection for trait Segregation Regeneration/ Selection for trait Final crop

10 Oligonucleotide directed mutagenesis (ODM) WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 10 Oligonucleotides - share homology with target sequence with the exception of the nucleotides to be modified - chimeric oligonucleotides (mixed DNA and RNA bases) - or single-stranded DNA oigonucleotides - 20 to 100 pb long Oligonucleotides target the homologous sequence in the genome Create one or more mismatch base pairs corresponding to the non-complementary nucleotides

11 Meganuclease technique WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 11 Meganucleases - Natural proteins acting as DNA-scissors - Recognise specific DNA sequence (12-30 bp) - Cause site-specific DNA break Possible applications as for ZFN technology

12 TALE WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 12 Transcription activator like effector (TALE) proteins - Proteins that bind to DNA in a sequence-specific way - Converted into DNA-scissors by binding to a nuclease (catalytic domain of FokI) - Can be modified for targeting a given sequence Possible applications as for ZFN technology Miller, J C, Nature Biotechnology 29, (2011)

13 Commercial pipeline WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 13 Zinc Finger Nuclease Technique (ZFN) Maize, oilseed rape and tomato research phase (trait not disclosed) phase III Workshop: First crops could be could be commercialized within 2-3 years. Oligonucleotide-directed mutagenesis (ODM) Oilseed rape, maize (herbicide tolerance) phase II to III Other main crops (trait not disclosed)

14 Commercial pipeline WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 14 Meganuclease technology: Crop in phase I PHASE I: Gene optimisation, crop transformation PHASE II: Trait development, pre-regulatory data, largescale transformation PHASE III: Trait integration, field testing, regulatory data generation (if applicable) PHASE IV: Regulatory submission (if applicable), seed bulk-up, pre-marketing Source: Lusser M. et al., New Plant breeding Techniques: State-of-the art and prospects for commercial development, JRC Technical Report EUR EN (2011)

15 Changes in the genome WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 15 Zinc finger nuclease 1 and 2 (ZFN-1 and -2) techniques ZFN-1 and -2 - ZFN gene expression transient - or in the case of gene insertion, progeny carrying the transgene is segregated out ZFN-1 - mutation in one or a few bp, short deletions or insertions (site-specific but random change) - mutation frequency varies, but usually rather low

16 Changes in the genome WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 16 ZFN-2 - Mutation in one or a few bp, short deletions or insertions (site-specific and specific change) - Mutation frequency varies, but usually rather low Possible unintended effects: ZFN-1 and -2 - Unintended mutations (e.g. because of lack of sequence specifity)

17 Changes in the genome WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 17 Oligonucleotide directed mutagenesis (ODM) Specific mutation (one or a few bp) Possible unintended effects: - Fewer unintentional changes expected than for breeding techniques based on irradiation and chemical mutagenesis Source: Glandorf B. et al., Evaluation of changes in the genome in plants through application of new plant breeding techniques in JRC Technical Report New plant Breeding Techniques, EUR EN

18 Detection/Identification WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 18 ZFN-1 and ZFN-2: PCR-based methods for the detection of small modifications (a single or few bp(s)) exist Provided information on nucleotide sequence (appr. 20 pb including the modification and its vicinity) is available, detection may be possible (specificity of primers?) However, identification not possible (not possible to distinguish from products obtained through other mutation techniques) Source: New plant breeding techniques: Challenges for detection and identification report of the New Techniques Task force (NTTF) in JRC Technical Report New plant Breeding Techniques, EUR EN (2011)

19 WORKSHOP COMPERATIVE SITUATION OF NEW PLANT BREEDING TECHNIQUES SEPTEMBER 2011 SEVILLE, SPAIN 19 Copyright: European Union, 2011