the rapid progress of crispr for targeted crop modificationtgc.ifas.ufl.edu/tbrt...
TRANSCRIPT
The rapid progress of CRISPR for targeted crop modification
47th Tomato Breeders Roundtable, 2018The Ohio State University, Wooster, Ohio
Andika Gunadi
Research Associate/PhD student
Advised by Dr. John J. Finer
Plant Transformation and Gene Expression Laboratory
The Ohio State University
1. Genome editing using CRISPR in plants
2. CRISPR in the news
3. Recent developments and applications
4. Our research
How do you modify your trait of interest?
• Marker-assisted breeding
• Untargeted mutagenesis– Radiation
– Chemicals
– Transposons
– Viruses
– Agrobacterium
– Biolistics
• Targeted mutagenesis– TALEN
– Zinc-finger nuclease
– Meganuclease
– CRISPR
DNA modification in plants
Nuclease(eg. Cas9)
Guide RNA(gRNA)
Targeted mutagenesis using CRISPR
Nuclease(eg. Cas9)
Guide RNA(gRNA)
2. Targeted DNA break
3. DNA repair
4. Plant recovery
5. Removal of transgenes
1. Introduction
Targeted mutagenesis using CRISPR
2. Targeted DNA break
3. DNA repair
4. Plant recovery
1. Introduction
Pros: - Targeted DNA modification- Broad target range- Time-efficient- Relatively cheap- Rapidly improving technology
DNA modification using CRISPR
5. Removal of transgenes
CRISPR in the news3294 publications in 20171135 in 2018 (NCBI-PubMed)
CRISPR is more versatile than other
genome-editing tools
CRISPR
TALENMeganuclease
ZFN
CRISPR in the news
Targeted knock-out of a polyphenol oxidase (PPO) gene in Agaricus bisporus, reducing the rate of browning, increasing shelf-life.
CRISPR in the news
Partial deletion of gene for amylose production in endosperm, saving several years of breeding into elite variety.
CRISPR in the news
Targeted mutation in the promoter region of
tomato genes
Generate useful trait variations
CRISPR in the news
cv. Moneymaker
Partial deletion of pathogen susceptibility gene
CRISPR in the newsUSDA does not regulate• “genetic deletions of
any size”• Targeted single base
changes• “insertion from
compatible plant relatives”
• “offspring of GE plant that does not retain the change of its parent”
Nuclease(eg. Cas9)
Guide RNA(gRNA)
2. Targeted DNA break
3. DNA repair
4. Plant recovery
5. Removal of transgenes
1. Introduction
Targeted mutagenesis using CRISPR
1. Introduction of CRISPR components
Particle bombardment Agrobacterium
Nuclease gene + gRNA gene
1. Introduction of CRISPR components
• No need for removing CRISPR components
• Useful for phenotypes that are easier to screen
• Currently lower efficiencies than DNA introduction
Generated indels at Liguleless1 (LIG), Acetolactate synthase (ALS2), and male fertility genes (MS26 and MS35)
2. Targeted DNA double-stranded break
Different Cas proteins require different target recognition sequence (PAM sites)
CRISPR Nuclease PAM Site(s) Reference
Cas9 NGG Bolotin et al. 2005, Microbiology
xCas9 NGG, NG, GAA, GAT Hu et al. 2018, Nature
Cpf1 TTTV Zetsche et al. 2015, Cell
Cpf1 variants TYCV, TATV Gao et al. 2017, Nature Biotechnology
Cms1 TTTN, GTTC Begemann et al. 2017, bioRxiv
2. Targeted DNA double-stranded break
Targeting several loci at once through multiplexing gRNA
3. DNA repair pathways and outcome
Indel
Large insertion
Large deletion
Donor DNA
Many publications on various strategies of genome editing
Single-base editing
4. Plant Recovery5. Removal of Transgenes
CRISPR requires plant transformation facility
Tomato is relatively easy to transform
• Screen for positive events
• Obtain homozygous T1 plants
• Cross to parental line for obtaining transgene-free genome-edited plants
Research at Finer laboratory
Soybean promoter characterization
Automated image collection
• CRISPR for targeted mutagenesis in promoters• CRISPR for targeted gene replacement
Transient transformation in
lima bean cotyledons
• Cas9 construct• gRNA construct• Parts for reconstituting GFP
No Cas9 CRISPR-mediated insertion
Reconstituted GFP
Research at Finer laboratory
Thank you for your attention
A tool is only as good as the hands that wield it
Plant Transformation LaboratoryDr. John J. FinerEric A. DeanHaiyan LiNina Ward Dept. of Horticulture
and Crop Science
2. Targeted DNA break3. DNA repair4. Plant recovery
1. Introduction
5. Removal of transgenes