Download Innovative Trait Development Tools in Plant Breeding will be Crucial

Innovative Trait Development Tools in Plant
Breeding will be Crucial for Doubling Global
Agricultural Productivity by 2050
Greg Gocal, Ph.D.,
Senior Vice President, Research and Development
CRISPR Precision Gene Editing Congress
Boston, MA
February 24, 2016
1.
Mission
To be leaders in the development of a
sustainable global non-transgenic food supply
2.
Cibus
Corporate Overview
•
Cibus is a privately owned biotechnology company with ~100 employees located in
San Diego and Minneapolis (US) and Kapelle (The Netherlands)
•
Cibus is primarily focused on the development of improved characteristics for crop plants
through the application of non-transgenic precision gene editing technology
•
Nucelis, an independent operating unit of Cibus, applies the same technology to develop
improved fermentation and bio-based products.
•
Cibus activities are funded through a combination of equity contributions and project
partnerships
Cibus Laboratories, San Diego, California
3.
Converging Technologies Creating a New Era in Agriculture
Genomics, Gene Editing & Cell Culture
Transgenic Era
New Era
Started in 1980’s
Started in 2000’s
Transgenic Insertions
(GMO Technology)
Advanced Breeding
(Non-transgenic)
In Agriculture
Total Addressable Market (TAM) is estimated
at 4B Acres (1.6B Hectares)
Hectares
(Million)
175.2
Transgenic technologies valued at $40B
yet have reached only 11% of the global TAM
~ 430M Acres /175M Hectares
Source: ISAAA
4.
Converging Technologies Have Enabled a New Non-Transgenic Industry
Cell
Culture
5.
Non-Transgenic Technology Path
RTDS™ GRON Explained
Creating a Change
GRON
1
A Gene Repair
Oligonucleotide (GRON) is
paired with the plant DNA
sequence. The pairing only
occurs at the gene target
region.
DNA
Strand
2
The GRON creates a
mismatch with the plant DNA
sequence.
3
Enzyme
4
6.
Following the repair, the GRON is
removed and the cell digests the
GRON. This is all part of the natural
process of cell division and
multiplication.
The plant’s native DNA
repair enzymes recognize
the mismatch and repair
the plant’s DNA using the
GRON as a template.
5
RTDS is complete and
the targeted gene has
been repaired.
What is the Rapid Trait Development System?
RTDS (Rapid Trait Development System) is a precision gene editing platform
Non-transgenic gene conversion platform
Highly precise, site-specific gene editing
Only the targeted base pair is changed
Does not leave behind foreign DNA
Works with the cell’s natural DNA repair
Oligonucleotide-directed mutagenesis
Successfully applied to multiple organisms
Recent technical advances have seen a
70-fold increases in efficiency.
7.
Gene Editing with Nucleases
Types of Nucleases with Varying Precision
Meganuclease
TALEN
*Adapted from: The Boston Consulting Group – Sep 2015
8.
Zinc-Finger
CRISPR-Cas9
DNA Double-Strand Break (DSB) Repair
DNA DOUBLE‐STRAND BREAKER
(Antibiotics, Meganucleases, ZFNs, TALENs and CRISPRs)
DSB
GENE DISRUPTION
GENE CORRECTION
GENE ADDITION
NHEJ
Conversion
Transgene
GRON ‐ NO
GRON ‐ YES
Sister Chromatid
GRONs enable precise sequence changes
9.
Precision Genome Editing in Flax
• GRONs and CRISPR/Cas9 were used to
develop an herbicide tolerance trait in
Linum usitatissimun (flax) by precisely
editing the 5’-enolpyruvylshikimate-3phosphate synthase (EPSPS) genes.
10.
Glyphosate Tolerance Utilizing EPSPS Gene
EPSPS
11.
Cell Biology in Flax
12.
Protoplasts
Microcalli
Calli
Shoots
Stage 1
Stage 2
Stage 3
Stage 4
(Single cell)
(100-1000 cells)
Delivery
Division
Calli Growth
Regeneration
Gene Editing Frequency Determined by NGS
Frequency of precise edits and indel scars as determined by deep
sequencing in three independent flax EPSPS editing experiments
Stage 2
13.
Stage 3
# of Calli
with
Targeted
Mutations
Exp #
Targeted
Mutation
Frequency
(%)
Indel
Mutations
(%)
# of Calli
Screened
1
0.23
19.8
5167
8 (0.15%)
2
0.10
19.2
4601
4 (0.08%)
3
0.09
19.6
NA
NA
Sequence Confirmation of Targeted Mutations
Callus with precise
edits
Sequence confirmation
14.
Regenerated plant
in soil
Targeted EPSPS Edits Provide Glyphosate Tolerance
15.
Segregation Analysis of C1 Plants
Targeted mutations show the expected 1:2:1 Mendelian
inheritance in the C1 generation
Genotype EPSPS Gene 2
16.
Plant ID
wt
Heterozygous
Homozygous
1
19
31
21
2
15
29
20
3
4
11
2
No Mutations Detected at Potential Off Target Sites
Off-target
ID
Sequence
# of
mismatches
Mutations Detected
Off‐1
ccgGTTACAGCAGCaGTCgGCGG
5
‐
Off‐2
ccgGTTACAGCAGCaGTCgGCGG
5
‐
Off‐3
tcaaaagCtGCAGCTaTCAGTGG
9
‐
Off‐4
tcaaaatCtGCAGCTGTCAGTGG
8
‐
Off‐5
tcaaaatCtGCgGCTGTCAGTGG
9
‐
Off‐6
tcaaaatCtGCgGCTGTCAGTGG
9
‐
Off‐7
aaggacACAGCAGCTGTCgGTGG
7
‐
Off‐8
accaaacgAGCAGCTGTCAGAGG
8
‐
On GCTGTTACAGCAGCTGTCAGCGG
0
+
Bases in red are mismatches with the On-target sequence
Analysis performed by amplicon deep sequencing
17.
No Integration of Plasmid in Converted Flax Plants
• C0 plants derived from callus A23 were analyzed for plasmid
integration by PCR
• Four sets of primer pairs each amplifying a different region of the
plasmid were used
A23
Plant 1
Plant 2
Plant 3
Plant 4
Plant 5
wt plant
Plasmid
Plasmid integration was not detected in any of the regenerated
plants
18.
Conclusions
• Precise targeted mutations were made in each flax EPSPS locus
• Isolated calli, with targeted mutations, were identified using molecular
screening alone
• Plants were regenerated from calli with the targeted mutations
• No unintended effects were observed
• The flax RTDS platform is primed for developing additional traits
19.
Thank you.
More information about RTDS and the
Cibus Team are presented at
www.cibus.com
20.