Project name:

SynPlast – understanding metabolic plasticity in synthetic biology

Research coordinator:

Sarah Agapito-Tenfen PhD (Norway, Scientist)

Research members:

Odd-Gunnar Wikmark PhD (Norway Scientist), Anne I. Myhr PhD (Norway, Director), Arinze Okoli PhD (Norway, Scientist),

Rubens Nodari PhD (Brazil, Professor), Miguel Pedro Guerra PhD (Brazil, Professor), Clarissa Caprestano PhD (Brazil, post-doc)

and Rodrigo Arnt Santanna (Brazil, undergrad student)

Institution:

GenØk - Center for Biosafety

Collaborative institution:

Federal University of Santa Catarina (Brazil)

Starting date:

June 2016

Duration:

12 months

Funding:

GenØk - Center for Biosafety

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Understanding metabolic plasticity in synthetic biology

Synthetic biology is a field of science and technology that still lacks an international agreed definition. In general, synthetic biology is perceived as the engineering or the re- engineering of biological components and systems in a novel way; a major development in genetic engineering and other biotechnological applications. Although it is a task that requires an intimate understanding of the biological process that is to be engineered, many products are already reaching commercial stages.

The classic example of synthetic biology is synthetic nucleases. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) is the most popular system these days due to its easy design. The potential applications of synthetic nucleases go well beyond simply creating mutations. These tools will find uses in various kinds of more comprehensive in vivo genome modifications (or even the construction of artificial genomes). Genome-editing techniques differ from those of classical genetic engineering because of their ability to (1) modify target genes in vivo, and not only in vitro followed by re- introduction; (2) increase the efficiency of introducing the intended modification at an intended place; and (3) increase the range of organisms in which the first two possibilities can be achieved.

Genome-editing techniques raise the possibility of targeting, in vivo, a specific gene or sequence in the genome of virtually any species. Targeted gene modification is the deletion, insertion or alteration of nucleotide order in an existing molecule of DNA or RNA. It is, however, also possible to insert or delete entire new genes or large sequences. Insertion of genes requires the supply of a DNA template along with the nuclease. However, confining the change to the intended template only is not possible. Thus, after the procedure, intended products are separated from unintended products. The current scientific knowledge about the safety of genome-editing techniques is based on a relatively small number of studies on tested genome-edited organisms and off- target sites. General conclusions would be premature at this point. The state of art about off-target activity of OGE, CRISPR, zinc-fingers, TALENs and meganucleases, and the corresponding knowledge gaps are provided in Agapito-Tenfen and Wikmark (2015 ) and in Agapito-Tenfen (2016).

Knowledge gaps remains in our understanding on how does CRISPR/Cas enzymes bind to DNA and DNA repair mechanisms, how does it search for the right target site, what are the off-target activities, how well is it tolerated in cells over long periods of time. Therefore, there is a pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any agricultural applications of CRSIPR/Cas9-mediated editing.

Therefore, this research project aims at understanding three biological aspects of CRISPR-edited plant cells: 1) efficiency and functioning of CRISPR system in plants; 2) potential off-target mutations and metabolic disturbances; 3) potential biosafety concerns related to metabolic changes. The project will develop a plant model to investigate target genomic modifications over time (cell division) – mutation stability, also the specificity of Cas9 to bind to gRNA – Cas9 sequence independent risks and indirect investigation of off-target effects by analyzing metabolic disturbance (omics analysis) – off-target ruling using naked delivery of Cas9 + gRNA into protoplasts of arabidopsis and maize species.

It is clear that the scientific knowledge about these new techniques is evolving, and as new information becomes available, knowledge gaps will no doubt diminish. However, it is also clear that proper regulation and mandatory risk assessment should be in place before genome-edited products are allowed on the market. It is crucial that regulators ask for experimental evidence to address potential adverse effects of genome-editing techniques in order to avoid a vacuum in the risk assessment of such organisms.

Below: Rodrigo Arnt Santanna presenting his undergrad work at Synplast for the Federal University of Santa Catarina student fair in August 2018 in Brazil.

Project publications

Agapito-Tenfen S.Z., Okoli A.S., Bernstein M.J., Wikmark OG., Myhr A.I. Revisiting Risk Governance of GM Plants: The Need to Consider New and Emerging Gene-EditingTechniques. Front Plant Sci. doi: 10.3389/fpls.2018.01874.

Agapito-Tenfen, S.Z. (2016). “Biosafety aspects of genome-editing techniques“, published by TWN – Third World Network and ACB. Policy Briefing November 2016.

Wikmark, O.-G., Brautaset, T., Agapito-Tenfen, S.Z., Okoli, A.S., Myhr, A.I., Binimelis, R. and Ching, L.L. (2016). “Synthetic biology – biosafety and contribution to addressing societal challenges“, Biosafety Report 02/16, GenØk-Centre for Biosafety, Tromsø, Norway.

Contact

Sarah Agapito-Tenfen (project coordinator)

Odd-Gunnar Wikmark

Photos taken by: Sarah Agapito-Tenfen, Clarissa Caprestano and Rodrigo Arnt Santanna.