Ingénierie cellulaire : amélioration et innovation technologiques pour les plantes d'une agriculture

Background

Prior to the start of GENIUS in 2012, genome editing had been considered as a potentially powerful complement to existing plant breeding methods, useful to answer upcoming challenges in agriculture, such as pesticide and input overuse, climate change or expectations for improved food. Genome editing appeared particularly attractive to ease the translation of biological knowledge into sustainable traits, thus enabling researchers and breeders to address these challenges through enhanced genetic progress, with more diverse, better adapted and yet high yielding plant varieties.

Objectives

The objectives stated for GENIUS in 2012 were to (i) implement innovative genome modification techniques in crops, (ii) improve the efficiency and throughput of crop transformation techniques, (iii) provide proof of concept for improved traits useful for a sustainable agriculture, (iv) acquire and maintain technical know-how and expertise in the domains of genetic and cellular engineering in France, (v) investigate gene flow from crop to weeds and (vi) assess the economic, ethical and legal impacts of novel plant breeding techniques. Eight years later, five of the six objectives were fully achieved (96% overall achievement rate for milestones, 93% for deliverables). Only the task focused on gene flow remained uncompleted, as it turned out difficult to produce the plant material required for the assessment.

To take into account the numerous technological developments in the field, and most notably the advent of CRISPR/Cas9 technology, the initial GENIUS work program was profoundly modified at several occasions. The relative simplicity and cost effectiveness of CRISPR/Cas9, in comparison to the initially foreseen meganuclease and TALEN technologies, liberated funding for additional tasks and allowed to address more diverse technological developments and biological targets, while respecting the project objectives.

Achievements

GENIUS (Genome ENgineering Improvement for Useful plants of a Sustainable agriculture) established in France genome editing technologies in 9 cultivated species (wheat, maize, rice, tomato, potato, oilseed rape, poplar, apple, rose) and 3 model species (Brachypodium, Physcomitrella, Arabidopsis). Replacing gradually meganuclease and TALEN approaches with CRISPR/Cas9 technology in the course of the project, targeted mutagenesis was achieved in all 12 species, and the more challenging gene editing by homology-based repair was completed in 3 of them. The recently emerged base editing alternative was implemented for 9 species. The partner labs also improved the efficiency of transgenesis in recalcitrant species (poplar, apple, rose) and for elite genotypes (maize, potato, apple, rose), thus facilitating the general use of genome editing in fundamental research and plant breeding. To illustrate the potential of genome editing in the transition towards a more sustainable agriculture, several proofs of concept were carried out in confined environments. The targets were disease resistance, early flowering, plant architecture, plant reproduction and salinity tolerance, traits useful to reduce pesticide use, adapt to climate change or shorten breeding cycles. Biological research was complemented with two social science studies. The first one mapped R & D networks in biotechnology and analysed the positioning of actors according to the commercial potential of the species under consideration, the second one documented a certain diversity of values and view-points, both within the GENIUS community and with farmers adopting alternative production systems.

Highlights

Mutant polQ is a booster of homologous recombination as concluded in a study showing that POLQ is involved in the NHEJ repair, acting as a homologous recombination repair inhibitor.

Early flowering in perennial species was triggered by inactivation of the MdTfl1.1 floral repressor in apple trees and overexpression of the flowering activator RoFT in rose.

Increase in petal number in rose (from 25–30 to 50–80) was achieved by reduced expression of AGAMOUS in Old Blush, an ancestor of modern roses.

In planta transformation in poplar was obtained by injection of A. tumefaciens into flower buds of several genotypes of Populus nigra, an indigenous species recalcitrant to in vitro transformation.

DFR, a visible marker system for targeted insertion was created in tomato; seedlings bearing targeted insertions were visually recognisable by a characteristic red rather than green colour.

Resistance of tomato to potyviruses was obtained by two targeted modifications in the eIF4E gene, required by potyviruses to carry out their infectious cycle.

Reduction of amylose content in potatoes was achieved by the simultaneous inactivation of all 4 alleles of the GBSS1 gene in potato.

Key numbers

GENIUS was funded for an 8-year period (2012-2020) by an aid of 6 M€ for a total cost of 21.6 M€. In this framework, 25 TALEN pairs (TALEN technology) and 266 guide RNAs (CRISPR-Cas9 technology) were transformed into plants, resulting in 2557 genome-edited plant lines with site-specific modifications. Including other approaches (amiRNA, genetic improvement of transformation frequencies), 363 DNA constructs were created and transformed, leading to 12,807 transgenic events. 49 articles were published in peer-reviewed journals and 5 in other publication supports. Project results were presented at 43 international and 59 national congresses. GENIUS results and materials yielded 35 collaborations and 16 MTAs signed with third party institutions.

Partnership

GENIUS was based on a public/private partnership that initially brought together 10 public and 5 private research teams from life science (12) and social science (3). Eight of the public partners were affiliated to INRAE, one to CIRAD and one to the University Lyon 3. The private partners were two biotechnology companies (Biogemma, Cellectis) and three plant breeding companies (Delbard, Germicopa and Vilmorin). Two partners left the project after completion of their tasks: Cellectis in 2016 and University Lyon 3 in 2018.

Beyond its tremendous scientific and technological progress, GENIUS was instrumental for the creation of a rich, technology-oriented community. Prior to the project, GENIUS scientists and engineers mainly worked in species-specific silos where developments in plant biotechnology were secondary to questions related to the biology of maize, tomato or other species. Today, they are active partners in a strong community where they exchange, discuss and further develop a shared know-how.

Impact

GENIUS provided temporary employment and high-level training for 61 young talents (postdocs, engineers, technicians) totaling approximately 770 person months. GENIUS scientists supervised 41 students (from BAC+2 to M2 levels), who discovered cutting edge genome editing technologies and forged their own opinion on their use through hands-on research.

Beyond academic and society-oriented publications, a patent was filed but did not result in licensing. The start-up company IAGE offering services in the field of genome editing was created by a former postdoc of the project.

The know-how in genome editing and plant transformation gained by GENIUS partners during the project allowed them to obtain additional funding for 29 related projects (total aid 5.3 M€). While GENIUS was technology-oriented, these spin-off projects generally addressed broader biological questions using GENIUS technology to generate new knowledge.

The impact for the private partners was very diverse and illustrated well different strategies, as genome-edited plants are still controversial and regulated as GMOs in Europe, contrary to other parts of the world. Cellectis transferred all agricultural biotechnology activities to its daughter company Calyxt in the USA. Delbard stopped investments in genome editing to focus on non-GMO research. In contrast, Biogemma, Germicopa and Vilmorin invested internally to further develop and apply the acquired know-how.

Finally, the GENIUS web site, brochures, leaflets and over 30 events (public debates, TV and radio interviews, press articles, stands) informed the general public about the project and provided fact-based information on genome editing.