Technologies du Futur Michel Caboche, Catherine Golstein, Gilles Pelsy

Technologies du Futur - 24 Novembre 2008

Technologies du FuturMichel Caboche, Catherine Golstein, Gilles Pelsy

INRA Paris, 24 Novembre 2008

A L I M E N T A T I O N A G R I C U L T U R E

E N V I R O N N E M E N T


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.


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


2. Consultation d’experts des technologies et champs d’application

– INRA / hors INRA– Contacts (collègues, réseau élargi, conférences)– Auteurs de publications– Recommandations (DS ou CD INRA)

-> Enrichir, orienter, corriger, valider les choix

Stratégie

1. Veille scientifique et technologique- Littérature scientifique- Rapports de projets de recherche- Conférences, colloques, workshops et séminaires- Sites internets variés- Etudes de prospective

-> Explorer les sciences et technologiesIdentifier, analyser, évaluer les technologies émergentes pertinentes

Ebauche de fiches technologiques


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


Sélection de technologies pertinentes pour TF

Technologie nouvelle?

Publication scientifique?

non oui

Potentiel d’application

atteint? non oui

Champ d’application pertinent pour l’agronomie?non oui

non oui

Fiche technologiqu

e

Fiche technologiqu

e

Technologie

Veille

(Association d’espèces)

(PCR)

(Single-Molecule Real-Time

Sequencing) (Recombinaison

homologue chez la souris)

Intérêt prolongé ou renouvelé


Standard technological worksheet1- Introduction, background2- Definition, description

How does it work? What makes it a new technology? What does it bring to previous technologies?

3- Current and prospective applicationsHow could it contribute to meet the needs of tomorrow’s agriculture?

4- Current limitations and challengesPotential technical limitations, risk assessments, ethical issues, research funding and management

5- Glossary of scientific terms6- Key references7- Consulted experts


Eventail de technologies émergentes et domaines d’application correspondants

Omics technologi

esGenetic

engineeringNanotechno

logiesImaging

technologiesPhenotyping 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 x x

Plant and animal

production systems

x x x x x x

Environment and industrial

ecologyx x x x x x

Feed and food x x x x x xNon-food and

green chemistry (industrial biotech)

x x x x x x

19 technologies, 7 catégories


Sensors, remote sensing and spatial analysisA 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

LAI, NDVI, sugar/acid

content

Plant vigor, N stress, maturity

Decision support

Agronomic models

European satellite Egnos, GPS


Sensors, remote sensing and spatial analysis

Potential applications:

Collecting informationsEnvironment monitoring,Nutritional status and epidemiology Surveillance of illicit cropsIdentification of management mistakes

Agronomic model feed with collected dataImprovement of model predictionsCrop management decision support

Precision farmingtracking and automatic guiding systems, variable-rate fertilisation water irrigation control in real timeCrop protection treatmentsOptimal harvest conditions

LimitationsCost and complexityPart 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


Landscape modelisationModelisation 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


Landscape modelisationPotential applications:Predictions on landscape evolution as a consequence of climatic changesOptimization of soil management (farming, natural areas, towns)Management of water ressources and biological invasionsManagement of the spreading of pathogens and creation of barriersManagement 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


High-throughput sequencing technologies

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)

Read length 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/Mbp50-fold cheaper than

Sanger~€5/Mbp

200-fold cheaper than Sanger

Limitations

- Expensive, - low-throughput, - labour intensive,

relatively short reads: issues with assembly of repetitive regions

Very short reads: issues for assembly or mapping and annotation.

- Accuracy limitations? GC -Short reads- Adoption of color space in sequence analysis?

Applications of choice

Method of choice for de novo sequencing of complex genomes

Best Sanger competitor for de novo sequencing projects for small and simple genomes and metagenomics projects.

Best for “seq-based” method analyses applied to sequenced genomes Best for resequencing projects, ultra deep SNP discovery and transcript.

Too few publications at this time to assess the range of SOLiD applications, and to compare its performance with competing technologies.

Three companies have developped new sequencing technologies (454, Illumina and SOLiD)that open multiple possibilities of applications. What is their respective interest?


Potential applications- GENOME LEVEL HTP de novo sequencingFacilitated 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 analysisDetection of rare somatic mutations (somaclonal variation)- TRANSCRIPTOME LEVELTranscript discovery and gene expression profilingAccurate 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)

LimitationsCost. Danger of simplistic views on research. Needs some thinkingConsulted experts : P Wincker, Génoscope

High-throughput sequencing technologies


High Throughput genotypingGenotyping 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


Genotyped loci

10

10

100

100

1,000

1,000

10,000

10,000

100,000

100,000

Gen

otyp

ed in

divi

dual

s

1,000,000

GWAS validation and

candidate gene association

Genome-Wide Association Studies

Plant and animal

breeding for selected traits

Candidate region fine mapping

Fingerprinting, Whole genome scans

Diagnostics

Applications enabled by HTP genotyping Diagnostics, MAS, disease related genes, Domestication traits, bar coding, industrial protection of genotypes


High Throughput genotyping techniques

Genotyped loci

10

10

100

100

1,000

1,000

10,000

10,000

100,000

100,000

Gen

otyp

ed in

divi

dual

s

1,000,000

GoldenGate assay

Infinium BeadChipsiselect

VeraCode GoldenGate

SNPlex,GenPlex

TaqMan Openarrays

iPLEX Gold

PyroseqSNaPshot

InvaderTaqMan

BeadChips

Illumina

AB

Sequenom

Targeted GeneChipsAffymetrix

Illumina High-Density 1M-Duo chipIlluminaAffymetrix Genome-Wide Human SNP Array 6.0

Genome-Wide Association Studies

Two main suppliers for GWA: ILLUMINA and AFFYMETRIX


Metagenomics

from the US National Academy of Sciences website http://dels.nas.edu/metagenomics/overview.shtml#process

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

-access to the uncultured (>99% bacteria)-access to whole microbe communities in a variety of natural environments (unlike pure cultures in artificial stable laboratory conditions)

Revolution in microbiology:

Enabled by newhigh-

throughput sequencing

technologies

http://dels.nas.edu/metagenomics/overview.shtml#process


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


Nutrigenomics and nutrigeneticsNutrigenomics: analyses the effects of nutrients and diets at the molecular and system’s levelEx: analysis of transcriptome/metabolome after Iron deprivation or the providing of phytosterols.

Nutrigenetics: analyses the effects of genetic makeup on individual responses to nutrients and diets

Ex: Identification of genotypes susceptible to obesity

Enabled by new

high-throuput ‘omics

technologies

Enabled by Human Genome Project, HapMap Project, and HTP

genotyping technologi

es

Association statistics of type 2 diabetes genome-wide association studies. From Frayling, Nat. Rev. Genet., 2007


Nutrigenomics and nutrigenetics applications and limits• Applications• Fundamental research: human and animal nutrition and health

– Better understanding of nutrition at the molecular level (mode of action of nutrients)– Identification of genetic loci predisposing to diet-related chronic disease– Identification of biomarkers associated with diet related chronic disease

• Feed/food industry:– Rational development and validation of claims on new functional feed/food products– Towards personalised nutrition: genetic testing-based diet recommendation?– Nutrition guidelines and market segmentation (ex Coeliac disease)

• Challenges• Challenges in genome-wide association analysis:

– large population, replication studies, population stratification, limitation to available SNPs…• Phenotyping challenges:

– For human subjects: uncontrollable heterogeneity of nutrition status and high costs• Challenges for genetics-based personalised nutrition

– 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:


Single molecule tracking

D Diffusion of a Glycin receptor tagged by a quantum dotBlue: outside synapse Green: inside synapse

1m

Single molecule track can be exploited to study the diffusion of macromolecules in the cell.They substitute a « real life » visualisationto a statistical view of processes

Single molecules can be visualised by bindinga chromophore (Ex GFP). Most chromophores bleachrapidly , preventing kinetics analysis. Their localization is limited by optical diffraction

Quantum dots can be substituted to chromophoresQD are issued from the chip technologyThey do not bleach and emit at specific wlThey can be localized at a precision of 10-20nmnot limited by optical diffraction.

Single molecules can also now be manipulated in the test tube to study their mechanical properties(Ex topoisomerases and supercoiled DNA)


Single molecule trackBenefitsMeeting of statistical physics, biochemistry and cytologyAccess to various molecular processesReceptor migration, endocytosis, cytoskeleton dynamics, etc…

LimitsSophisticated techniques that need optical engineeringNot commercially availableQD still sterically big, may create artifacsTracking the interaction of two different molecules is a challenge

Consulted experts: B. Satiat Jeunemaitre, CNRS Gif, A Triller, ENS Ulm


Creation of novel enzymatic activities

-Combination rational design / directed evolution-Computational design based on crystal structure or homology modeling, and phylogenetic analysis

10,000 fold improvement in catalytic efficiency

Example: DNA shuffling of GAT

-> Novel/improved biocatalysts for chemical, food and pharmaceutical industries

Itera

tive

roun

ds

(After Johannes and Zhao, 2006)

Random/targeted mutagenesis or Gene shuffling

Selection or HTP screening

Novel substrate specificity or

Improved catalytic activity

Target gene(s)

Goal achieved

Library of mutant genes

Library of mutant enzymes

Functionally improved enzymes

The diversity of X-rays establishedenzyme 3D structures seem to reach a plateau. How to create diversity?

Goal:Creation of new enzyme specificities by active site random mutagenesis and htp test of these activities on new substrates is a novel avenue


Potential applicationsDesign of novel enzymatic activities and analysis of

the basis of substrate specificty.Creation of enzymes working on artificial substratesPotential applications in microbiology/fermentation/ second generation of biofuels /remediation

LimitationsEnzyme 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, FranceMichael O’Donohue, INSA, Toulouse, France

Creation of novel enzymatic activities


Targeted gene modification/inactivationHomologous 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 technologyTILLING 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 technologyonce 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.


Targeted gene modificationPotential applications of TILLING, RNAi, ZFN and Meganucleases

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

RNAiThe technique relies on the production of transgenics. No expected toxicityThe 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 MeganucleasesMore performant than TILLING and RNAi as regards the diversity of target modifications. New therapies against DNA virus infectionClaimed 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


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


Induced pluripotent stem cells Potential applicationsBasic 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 diseasesFarm animal engineering. Despite numerous efforts, pluripotent ES cells from farm animals have not been obtained. This is strongly limiting the production of trangenic animals

LimitationsThe 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


Ecological intensification

Phenotyping technologies

Synchrotron beams

Mass spectrometry, Proteome, Metabolome

Nanotechnologies

Synthetic biology


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


Merci de votre attention

A L I M E N T A T I O N A G R I C U L T U R E

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Documents

Technologies du Futur Michel Caboche, Catherine Golstein, Gilles Pelsy