Transcript Technologies du Futur

Technologies du Futur
Michel Caboche, Catherine Golstein, Gilles Pelsy
INRA Paris, 24 Novembre 2008
Technologies du Futur - 24 Novembre 2008
ALIMENTATION
AGRICULTURE
ENVIRONNEMENT
Lettre de mission
Technologies du Futur
Contexte:
•
« (…) l’INRA doit être à même d’anticiper et de susciter les grandes
ruptures scientifiques et technologiques susceptibles de survenir dans les
décennies à venir et d’avoir un impact fort sur l’agriculture et
l’alimentation. »
Mission:
•
« (…) compléter les travaux d’Agrimonde* par une étude spécifique sur les
nouvelles technologies et leur importance en recherche agronomique, sur
un champ suffisamment large pour y inclure la biologie, l’écologie, les
sciences agronomiques, et les technologies de transformation et de suivi
de la qualité. »
Agrimonde*: prospective INRA/CIRAD sur les systèmes agricoles et
alimentaires mondiaux à l’horizon 2050.
Technologies du Futur - 24 Novembre 2008
Objectif
• Identifier et analyser des technologies émergentes
– pertinentes pour la recherche agronomique
• la biologie
• l’écologie et les sciences agronomiques
• la transformation et qualité des produits alimentaires et non-alimentaires
– susceptibles de répondre aux enjeux de l’agriculture de demain
• Délivrables:
– Etablissement d’une liste d’une douzaine de technologies
émergentes
– Séminaire de réflexion / validation/élargissement
– Collection de fiches technologiques
– Rapport final résumant les conclusions de l’étude
Technologies du Futur - 24 Novembre 2008
Stratégie
1.
Veille scientifique et technologique
->
Explorer les sciences et technologies
Identifier, analyser, évaluer les technologies émergentes pertinentes
Ebauche de fiches technologiques
2.
Consultation d’experts des technologies et champs d’application
->
Enrichir, orienter, corriger, valider les choix
-
–
–
–
–
Littérature scientifique
Rapports de projets de recherche
Conférences, colloques, workshops et séminaires
Sites internets variés
Etudes de prospective
INRA / hors INRA
Contacts (collègues, réseau élargi, conférences)
Auteurs de publications
Recommandations (DS ou CD INRA)
Technologies du Futur - 24 Novembre 2008
Les nouvelles technologies ainsi que leurs
perfectionnements conduisent a de nouvelles
découvertes et de nouvelles inventions
Leur émergence est un phénomène assez rare,
souvent fortuit
Rien ne permet de penser que ce processus
ralentisse
Technologies du Futur - 24 Novembre 2008
Sélection de technologies pertinentes pour TF
Technologie
Technologie nouvelle?
Intérêt
prolongé ou
renouvelé
non
Potentiel
d’application atteint?
non
oui
Publication scientifique?
non
(PCR)
(Association d’espèces)
Fiche
technologique
oui
(Single-Molecule
Real-Time
Sequencing)
Veille
Technologies du Futur - 24 Novembre 2008
oui
Champ d’application
pertinent pour l’agronomie?
(Recombinaison
homologue
chez la souris)
non
oui
Fiche
technologique
Standard technological worksheet
1- Introduction, background
2- Definition, description
How does it work? What makes it a new technology? What does it
bring to previous technologies?
3- Current and prospective applications
How could it contribute to meet the needs of tomorrow’s
agriculture?
4- Current limitations and challenges
Potential technical limitations, risk assessments, ethical issues,
research funding and management
5- Glossary of scientific terms
6- Key references
7- Consulted experts
Technologies du Futur - 24 Novembre 2008
Eventail de technologies émergentes et
domaines d’application correspondants
Omics
technologies
Genetic
engineering
Nanotechnolog
ies
Imaging
technologies
Phenotyping
technologies
Agronomy and
agricultural
production
technologies
Bioinformatics
and
computational
tools
Research in
biology
x
x
x
x
x
x
x
Plant and animal
breeding
x
x
Plant and animal
production systems
x
x
x
x
x
x
Environment and
industrial ecology
x
x
x
x
x
x
Feed and food
x
x
x
x
x
x
Non-food and
green chemistry
(industrial biotech)
x
x
x
x
x
x
x
x
19 technologies, 7 catégories
Technologies du Futur - 24 Novembre 2008
Sensors, remote sensing and spatial analysis
A variety of wireless sensors for monitoring biomass, soil and fruit quality, combined with spatial
analysis can feed agronomic models in real time and help rapid decision making at the farm level.
A high GPS resolution is needed. The Egnos satellite coupled with on site beacons will provide
a less than 1 meter resolution
European satellite
Egnos, GPS
Plant vigor,
N stress,
maturity
Agronomic
models
Decision support
Technologies du Futur - 24 Novembre 2008
LAI, NDVI,
sugar/acid
content
Sensors, remote sensing and spatial analysis
Potential applications:
Collecting informations
Environment monitoring,
Nutritional status and epidemiology
Surveillance of illicit crops
Identification of management mistakes
Agronomic model feed with collected data
Improvement of model predictions
Crop management decision support
Precision farming
tracking and automatic guiding systems,
variable-rate fertilisation
water irrigation control in real time
Crop protection treatments
Optimal harvest conditions
Limitations
Cost and complexity
Part of the collection of data is still hand-based (ex Spectrophotometer)
Present GPS resolution is limiting. Cloudy areas are a problem
Consulted experts: Jean-Michel Roger, Bruno Tisseyre, Alexia Gobrecht
Technologies du Futur - 24 Novembre 2008
Landscape modelisation
Modelisation is a basic tool for many aspects of agronomy sciences. Models have been set
up to handle the complexity of the development of a crop, or to describe a cropping system
and its management at the field level. The management of water ressources in a river basin
can also be modelized. But for the evaluation of the socio-economic and environmental
consequences of a decision (ex: Large scale biofuel production) these different levels
have to be integrated.
Landscape modelisation which aims at integrating these different levels from the plant to the
ecosystem is a challenging research domain which has potentially important applications.
AMAP modelisation
of the consequences
of climatic changes
on the landscape
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Landscape modelisation
Potential applications:
Predictions on landscape evolution as a consequence of climatic changes
Optimization of soil management (farming, natural areas, towns)
Management of water ressources and biological invasions
Management of the spreading of pathogens and creation of barriers
Management of GMO / organic farming
Optimization of farming practices as a consequence of market evolution
Limitations:
Models are still far from integrating the desired informations.
They are not ofte interoperable
New modelisation tools need to be set up, reducing
the size of complex objects and managing the heterogeneity of data
Consulted experts: F. Garcia, J. Wery, H. Descamps
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High-throughput sequencing technologies
Three companies have developped new sequencing technologies (454, Illumina and SOLiD)
that open multiple possibilities of applications. What is their respective interest?
ABI 3730XL
(Applied Biosystems)
(released in 2005)
GS FLX System
(Roche Diagnostics)
(released in October
2005)
Illumina Genome
Analyzer
(Illumina)
(released in June 2006)
SOLiD DNA Sequencer
(Applied Biosystems)
(released in October 2007)
600-900 bp
400 bp
18, 26, 36 bp
35 bp
Data per
run
1 Mbp
500 Mbp
1.5 Gbp
(3 Gbp for mate pairs)
4 Gbp
(8 Gbp for mate pairs)
Machine
cost
€400,000
€ 450,000
€500,000
€ 550,000
Run cost
per base
~€1000/Mbp
€20/Mbp
50-fold cheaper than
Sanger
Read
length
Limitations
Application
s of choice
- Expensive,
- low-throughput,
- labour intensive,
Method of choice for de
novo sequencing of
complex genomes
relatively short reads:
issues with assembly of
repetitive regions
Best Sanger competitor
for de novo sequencing
projects for small and
simple genomes and
metagenomics projects.
Technologies du Futur - 24 Novembre 2008
~€5/Mbp
200-fold cheaper than Sanger
Very short reads: issues
for assembly or mapping
and annotation.
Best for “seq-based”
method analyses applied
to sequenced genomes
Best for resequencing
projects, ultra deep SNP
discovery and transcript.
- Accuracy limitations? GC
-Short reads
- Adoption of color space
in sequence analysis?
Too few publications at
this time to assess the
range of SOLiD
applications, and to
compare its performance
with competing
technologies.
High-throughput sequencing technologies
Potential applications
- GENOME LEVEL
HTP de novo sequencing
Facilitated sequencing of related genomes
Resequencing
genetic diversity and evolutionary studies:
genome-wide discovery of genetic polymorphisms
Low-cost alternatives to whole-genome resequencing:
Environmental sequence analysis
Detection of rare somatic mutations (somaclonal variation)
- TRANSCRIPTOME LEVEL
Transcript discovery and gene expression profiling
Accurate gene annotation ( splicing sites)
Deep sequencing of small RNAs (miRNA, si RNAs, etc)
DNA Methylation profiling (BS-seq, methylC-seq)
ChIP sequencing: DNA protein binding sites analysis Epigenome analysis
( nucleosome mapping, analysis of epigenetic processes, etc)
Limitations
Cost. Danger of simplistic views on research. Needs some thinking
Consulted experts : P Wincker, Génoscope
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High Throughput genotyping
Genotyping and phenotyping are at the root of genetic analysis .
Phenotyping can be performed on multiple, unrelated criteria,
All genotyping technologies exploit the variations occurring in genomic
sequences. The same techniques can be used to study human genetics
but also cattle and crop genetics.
Among DNA polymorphisms, SNP variations are found in all genomes and
can be identified by exploitation of htp genome re-sequencing.
A large number of SNP detection/genotyping techniques are available.
What’s new? Genome-wide association for LD analysis.
Rationale: The genes contributing to a specific trait are difficult to predict.
Its more efficient to scan them by detection of associations, using a dense array
of SNPs (Ex: an average of 10 SNPs per gene and a total of 1000 000 SNPs
per human genome) Two main suppliers: ILLUMINA and AFFY.
Limitations: only one or two individuals analysed on one array. Large numbers
(1000 genotypes) required for statistical significance. Problems of population
structures. Very expensive…
Consulted experts: A. Eggen, D. Brunel, A. Charcosset, I. Gut
Technologies du Futur - 24 Novembre 2008
Applications enabled by HTP genotyping
Diagnostics, MAS, disease related genes, Domestication traits,
bar coding, industrial protection of genotypes
Genotyped individuals
100,000
10,000
1,000
Plant and
animal
breeding for
GWAS
selected traits
validation and
candidate gene
association
Candidate region
fine mapping
Genome-Wide Association Studies
100
Diagnostics
Fingerprinting, Whole genome scans
10
10
100
1,000
10,000
Genotyped loci
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100,000
1,000,000
High Throughput genotyping techniques
Two main suppliers for GWA: ILLUMINA and AFFYMETRIX
Genotyped individuals
100,000
Genome-Wide Association Studies
10,000
iPLEX
Gold
1,000
Sequenom
SNPlex, AB
TaqMan
Invader GenPlex
SNaP
Illumina
100 shot Pyroseq
GoldenGate
VeraCode
TaqMan
assay
GoldenGate
Openarrays
10
Illumina High-Density 1M-Duo chip
Affymetrix Genome-Wide Human SNP Array 6.0
Illumina
iselect
Infinium BeadChips
Affymetrix
Targeted GeneChips
10
100
1,000
10,000
Genotyped loci
Technologies du Futur - 24 Novembre 2008
100,000
BeadChips
1,000,000
Metagenomics
Metagenomics (whole-community genomics or environmental genomics) : field of study of the
metagenome, defined as all the genomes of a microbe community in a particular environment.
Metagenomics helps preforming the inventory of living organisms in a specific biotope
Enabled by new
high-throughput
sequencing
technologies
from the US National Academy of Sciences website http://dels.nas.edu/metagenomics/overview.shtml#process
-access to the uncultured (>99% bacteria)
Revolution in microbiology: -access to whole microbe communities in a variety of natural environments
(unlike pure cultures in artificial stable laboratory conditions)
Technologies du Futur - 24 Novembre 2008
Metagenomics applications
** Microbes ubiquitous and essential to life
• Fundamental research: microbe diversity and evolution, ecology,
biology
• Environment: Biosensors, bioremediation of contaminated soils,
industrial treatment of wastewater
• Agriculture: Optimisation of natural plant fertilisation, rapid
identification of pathogens responsible of emerging diseases
• Human nutrition and health: Search for new antibiotics, role of
human gut microbes (microbiome) in nutrition and obesity
• Bioindustry: discovery of novel enzymatic activities (ex. enzymes
specialised in lignocellulose degradation in termite guts)
Consulted experts on Metagenomics:
•Dusko Ehrlich, Génétique microbienne, INRA Jouy-en-Josas, France
•Denis Le Paslier, Génoscope, Evry, France
•Jean Weissenbach, Génoscope, Evry, France
•Pierre Monsan, INSA, Toulouse, France
•Michael O’Donohue, INSA, Toulouse, France
•Renaud Nalin, LibraGen, Toulouse, France
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Nutrigenomics and nutrigenetics
Nutrigenomics: analyses the effects of nutrients and diets at the molecular and system’s Enabled by new
level
high-throuput
Ex: analysis of transcriptome/metabolome after Iron deprivation or the providing of
‘omics
phytosterols.
technologies
Nutrigenetics: analyses the effects of genetic makeup on individual responses to nutrients
and diets
Ex: Identification of genotypes susceptible to obesity
Enabled by
Human
Genome
Project,
HapMap
Project, and
HTP
genotyping
technologies
Association statistics of type 2 diabetes genome-wide association studies. From Frayling, Nat. Rev. Genet., 2007
Technologies du Futur - 24 Novembre 2008
Nutrigenomics and nutrigenetics applications and limits
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Applications
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Fundamental research: human and animal nutrition and health
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Better understanding of nutrition at the molecular level (mode of action of nutrients)
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Identification of genetic loci predisposing to diet-related chronic disease
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Identification of biomarkers associated with diet related chronic disease
Feed/food industry:
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Rational development and validation of claims on new functional feed/food products
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Towards personalised nutrition: genetic testing-based diet recommendation?
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Nutrition guidelines and market segmentation (ex Coeliac disease)
•
Challenges
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Challenges in genome-wide association analysis:
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•
Phenotyping challenges:
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•
large population, replication studies, population stratification, limitation to available SNPs…
For human subjects: uncontrollable heterogeneity of nutrition status and high costs
Challenges for genetics-based personalised nutrition
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Validation of relationship between genetic marker and health status?
–
Genetic tests miss extra sources of variability: the environment, the epigenome,etc
Consulted experts on nutrigenomics and nutrigenetics:
Technologies du Futur - 24 Novembre 2008
Single molecule tracking
Single molecule track can be exploited to
study the diffusion of macromolecules in the cell.
They substitute a « real life » visualisation
to a statistical view of processes
Single molecules can be visualised by binding
a chromophore (Ex GFP). Most chromophores bleach
rapidly , preventing kinetics analysis. Their
localization is limited by optical diffraction
1mm
Quantum dots can be substituted to chromophores
QD are issued from the chip technology
They do not bleach and emit at specific wl
They can be localized at a precision of 10-20nm
not limited by optical diffraction.
D
Single molecules
can also now be manipulated in the
test tube to study their mechanical properties
(Ex topoisomerases and supercoiled DNA)
Technologies du Futur - 24 Novembre 2008
Diffusion of a Glycin receptor
tagged by a quantum dot
Blue: outside synapse
Green: inside synapse
Single molecule track
Benefits
Meeting of statistical physics, biochemistry and cytology
Access to various molecular processes
Receptor migration, endocytosis, cytoskeleton dynamics, etc…
Limits
Sophisticated techniques that need optical engineering
Not commercially available
QD still sterically big, may create artifacs
Tracking the interaction of two different molecules is a challenge
Consulted experts: B. Satiat Jeunemaitre, CNRS Gif, A Triller, ENS Ulm
Technologies du Futur - 24 Novembre 2008
Creation of novel enzymatic activities
The diversity of X-rays established
enzyme 3D structures seem to reach
a plateau. How to create diversity?
Example: DNA shuffling of GAT
Goal:
Creation of new enzyme specificities
by active site random mutagenesis and
htp test of these activities on new
substrates is a novel avenue
Iterative rounds
Target gene(s)
Random/targeted mutagenesis or
Gene shuffling
Library of mutant genes
10,000 fold improvement in catalytic efficiency
Library of mutant enzymes
Selection or HTP screening
Functionally improved enzymes
Novel substrate specificity
or
Improved catalytic activity
Goal achieved
(After Johannes and Zhao, 2006)
Technologies du Futur - 24 Novembre 2008
-Combination rational design / directed evolution
-Computational design based on crystal structure
or homology modeling, and phylogenetic analysis
-> Novel/improved biocatalysts for chemical, food
and pharmaceutical industries
Creation of novel enzymatic activities
Potential applications
Design of novel enzymatic activities and analysis of
the basis of substrate specificty.
Creation of enzymes working on artificial substrates
Potential applications in microbiology/fermentation/
second generation of biofuels /remediation
Limitations
Enzyme design requires a large set of competences to
be operational (3D protein analysis, modelisation, site
directed mutagenesis, htp screen for the detection of
improved enzyme
Consulted experts :
Pierre Monsan, INSA, Toulouse, France
Michael O’Donohue, INSA, Toulouse, France
Technologies du Futur - 24 Novembre 2008
Targeted gene modification/inactivation
Homologous recombination is used in model organisms to perform targeted gene modification.
However it does not work on most species. Four technologies can partially substitute.
TILLING is a methodology that allows the screen of mutations affecting a gene of interest in
large populations of plants issued from a mutagenic treatment. It’s a well established technology
RNAinterference is a process of gene inactivation induced by the recognition by the cell
machinery of short (20-23nt) double stranded RNAs. It has a high sequence selectivity. It’s
exploited through GM technology
TILLING and RNAi are well established technologies but recent developments are promizing
Zinc Finger Nucleases (Sangamo biosciences, Ca) are able to recognize and cut a specific DNA
sequence. They act as dimers of three zinc finger proteins that recognize a set of 3X3 nt linked
to an endonuclease, leading to a specificity of 18 nt (14 in fact).
Meganucleases (Cellectis SA) are restriction enzymes with
very high sequence selectivity. They can be engineered
in heterodimeric endonucleases in which
a set of 8 aai recognize seven bases in the DNA target
leading to a specificity of 2X7 nt. As for ZFN technology
once a double strand break is generated, NHEJ repair
generates mutations at the site. Meganucleases can be
used for gene exchange by deleting a fragment of up to
8kb and replace it by a new version. In both technique a
combinatorial screen for ZFN or Mega nucleases of
desired specificity is necessary.
Technologies du Futur - 24 Novembre 2008
Targeted gene modification
Potential applications of TILLING, RNAi, ZFN and Meganucleases
RNAi
Reverse genetics and targeted gene inactivation
(Ex creation of recessive resistances to viruses with TILLING)
Inactivation by KO of genes with undesirable effects (ex: synthesis of alkaloïds
Induction of allelic diversity to optimize an agronomic trait
The technique relies on the production of transgenics. No expected toxicity
The production of a Si RNA by the host can lead to the inactivation of
an essential gene in a pest that feeds on this host, resulting in protection
of the plant against the pest.
Potential applications of ZFN and Meganucleases
More performant than TILLING and RNAi as regards the diversity
of target modifications. New therapies against DNA virus infection
Claimed targeted introduction of genes (Ex: to cure a disease)
Limitations
ZNF and Meganuclease technologies are work intensive and expensive
The ZFN technology does not work well in different labs.
Consulted experts: A. Choulika, Cellectis; B. Dujon, Pasteur
Technologies du Futur - 24 Novembre 2008
Induced pluripotent stem cells
The regeneration of an organism from one of its
somatic cells can be achieved in the plant kingdom,
but generally not in the animal kingdom, including
mammals.
This restricts the exploitation of cell therapy
approaches as well as gene transfer techniques to
embryogenic stem cells which are not easy to
handle.
iPS (Induced pluripotent stem cells) provide a
breakthrough in this field by dedifferentiating
somatic cells and reinitiating a pluripotent state.
This is achieved by the transfection of several
transcription factors that trigger the process of
epigenetic reprogramming. Such reprogrammed cells
injected in blastocysts lead to viable animals
Technologies du Futur - 24 Novembre 2008
Induced pluripotent stem cells
Potential applications
Basic research. Understanding the basis of cell differenciation…
Cell therapy. A fibroblast cell issued from an animal carrying a genetic
disease can be cured of the mutation by gene transfer, and then
converted into an iPS cell that can be differentiated, upon proper
stimulation in a specific type of stem cell susceptible to colonize the
Diseased animal, in the absence of non self rejection.
Numerous applications foreseen to cure human genetic diseases
Farm animal engineering. Despite numerous efforts, pluripotent ES cells
from farm animals have not been obtained. This is strongly limiting the production of
trangenic animals
Limitations
The technique of transfection of the cocktail of four transcription factors is based
on retroviral vectors. This can unpredictably generate tumor formation.
This could be alleviated by transient expression of the introduced TF that dont
need to be permanently expressed to confer the iPS phenotype
Consulted experts: JP Renard, INRA
Technologies du Futur - 24 Novembre 2008
Ecological intensification
Phenotyping technologies
Synchrotron beams
Mass spectrometry, Proteome, Metabolome
Nanotechnologies
Synthetic biology
Technologies du Futur - 24 Novembre 2008
Séminaire de validation
•
Paris, 23 Janvier 2009
•
Participants sur invitation :
– DS INRA/CIRAD, chercheurs consultés et additionnels, public 64
invitations/privé 69 invitations, toutes disciplines
•
Objectifs :
• Evaluer de la pertinence des choix des technologies sélectionnées
• informer les participants sur les technologies émergentes
• susciter des décloisonnements de champs d’application
• susciter des pistes d’applications non-envisagées,
•
Modalités
– Une session pleinière
• présentation de l’étude/ analyse par trois experts de ce qui bouge dans leur domaine
– Trois groups de travail
• Biologie + Agronomie; Agronomie+ Transfo et Qualité; Transfo et Qualité+ Biologie
– Travail de synthèse
• Sur place: conclusions des trois groupes
• Les participants seront convié a transmettre leurs remarques aux organisateurs
Technologies du Futur - 24 Novembre 2008
Merci de votre attention
Technologies du Futur - 24 Novembre 2008
ALIMENTATION
AGRICULTURE
ENVIRONNEMENT