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If we obtain a higher expression level of the weak promoter with our two tools, it could lead to several useful applications. For example, we would be able to use this tool to enhance gene expression of any endogenous genes due to CRISPR/Cas9 specificity. Indeed, it would be possible to design specific sgRNA to target specific sequences. Our solution leads to several advantages.

Study the chromosome structure and function

During our bibliographic research in order to build the project and write the background part of the project, it appeared to us that research works on chromosome structure and function in bacteria were rare and very specialized. During the first months, we have met Dr. Olivier Espéli, researcher in chromosome dynamics at the Collège de France. During this meeting, it appeared that the tool we wanted to design could be very useful for research on the same topic, as it is also mentioned in a paper submitted by Olivier Espéli:

The most important challenge is the development of selectable reporters for chromosome structural properties. Consider, for instance, the problem of a selection that depends on the contact between two arbitrary genomic loci. One possible solution to this problem is to adapt a protein-fragment complementation assay (PCA). In a classic PCA assay, two proteins of interest (targets) are genetically linked to two moieties of an enzyme (reporter) that can confer resistance to the bacterium. Because the reporter is fully assembled only when the two target proteins interact, the fraction of surviving bacteria after an antibiotic treatment provides quantitative information regarding the interaction energies between the two proteins. We surmise that this assay may be naturally transposed to the problem of the contact between two genomic loci. To this end, instead of considering two target proteins, we propose to consider a unique target that can recognize two genomic loci. For example, the CRISPR/Cas9 system with two different short sgRNAs could be used to direct the target, a modified Cas9 with no endonuclease property, to the genomic loci. The modified Cas9 would then be used in two different fused systems, each one containing one reporter moiety [Fig. 1]. A possible protocol would then consist of three stages: (i) activation of the CRISPR-Cas9-PCA system, (ii) antibiotic treatment, and (iii) sequencing of the survivors (Lagomarsino et al., 2015).

For chronological purpose, we would like to precise that we had the idea of using CRISPR/Cas9 system before the meeting, and this meeting enabled us to precise our project choice, by the interest for famous researcher to use our tool.

Enhance the expression of any genes: epigenetic engineering

The global goal of our project is to study the impact of the spatial organization of the chromosome on the genetic transcription. For that purpose, we designed a tool to characterize the effect of a strong promoter on a weak promoter in space. But in a way, we hope that the fact that the weak promoter is spatially close to the strong one would influence and increase (so induce) it strength. This enhancement of the transcription of the gene controlled by the weak promoter could be very interesting in many fields and could be used for research and in the industry. Indeed, promoter strength, or activity, is important in genetic engineering and synthetic biology. Furthermore, this enhancement is based on the chromosome organization, the tool we designed correspond to an ‘’epigenetic’’ engineering tool.

In order to increase the expression of a specific gene on the bacterial chromosome, a classic method is to choose one promoter with a suitable strength which can be employed to regulate the rate of transcription, which then leads to the required level of protein expression. For this purpose, so far, many promoter libraries have been established experimentally (Li & Zhang, 2014). Then the strategy is to clone the chosen promoter just before the gene sequence [Fig. 2a]. With our tool, we offer a new strategy which also involves a cloning (the plasmid of interest) but no insertion in the bacterial chromosome, which is an advantage!

Instead of choosing a promoter, the user has to chose an endogenous strong promoter on the bacterial chromosome and has to know it sequence. He also has to know the sequence of the gene he wants to enhance the expression. Knowing these sequences, the user will be able to design the two sgRNAs and then construct the expression plasmid. Several resources exist on internet in order to design the sgRNA (Kim & Kim, 2014) by bio-informatics. The strains are then transformed with the plasmid containing our tool, and this plasmid will be expressed ‘’in vivo’’, enabling the enhancement of the weak promoter [Fig. 2b]. Compared to the usual strategy, it seems longer because of the bio-informatics but at the end, our tool presents several advantages, described as follow.

For research

Our tool is composed of parts available on the iGEM Registry and ‘’as part of the synthetic biology community's efforts to make biology easier to engineer, it provides a source of genetic parts to iGEM teams and academic labs’’. So our tool will be available for all Research Institute which wants to use it, and we think it will be the case because we have met some scientists from I2BC and the Pasteur Institute who seems very interested in.</pr>

For the industry

Intellectual property

In our system, we use the CRISPR/Cas9 technology and FRB/FKBP12 system, so we have to be aware of intellectual property issues.

The CRISPR/Cas9 technology is now free to use, as there is an issue in order to know which team patented first. The patent race opposes several teams but there are two major players in the battle to secure rights to the CRISPR/Cas9 system: the group headed by Jennifer Douda from the University of California and the group headed by Feng Zhang from the Broad Institute of Harvard and MIT. While Doudna’s group may have published first and have received a number of awards for their work in this field, it is Zhang who has been awarded the first patent on the basic CRISPR/Cas9 technology – US Patent No. 8.697.359. The final decision is due for 2017 (Smith-Willis & San Martin, 2015).

The FRB/FKBP12 system is sold by several companies as CloneTech for instance and the system is subject to some patents, as patent US6187757 B1 for instance.

===Compatibility of our system with the industry

The compatibility issue of our system with the industry and the industrial scales seems to be the main issue. In the field of white biotechnologies (meaning industrial biotechnologies), it exists a lot of improved stains in order to produce economically speaking interesting compounds. Two ways exist to produce an interesting compound by a biological way (most of time bacteria or yeast):

• Bioconversion: the conversion of one chemical compound, or one form of energy, into another by living organisms
• ‘’De novo’’ synthesis: synthesis of complex molecules from simple molecules such as sugars or amino acids

There are many advantages for a company to produce compounds by micro-organisms: it is generally cheaper (when the process is controlled) and eco-friendlier that compounds produced by chemical way. Furthermore, the strains used are produces by genetic engineering and more and more by synthetic biology. A lot of the genetic engineering work consists of an improvement of the expression of some specific genes, which could be done also with out tool. The wide majority of these strains are GMOs as they own exogenous (meaning foreign) DNA on their genome. Once a company has the good strain (after many years and several million dollars), the strain is introduced in a big fermenter.

It exists now a lot of examples of such compounds produced by biological way but we focused ourselves on one specific example. In recent years, biotechnology-derived production of flavors and fragrances has expanded rapidly. The world's most popular flavor, vanillin, is no exception. Thus, it exists a wide range of biotechnology-based vanillin synthesis with the use of ferulic acid, eugenol, and glucose as substrates and bacteria, fungi, and yeasts as microbial production hosts (Gallage and Møller, 2015).

Our system seems to be a good solution for synthetic biology, but the expression of our tool via the plasmid previously inserted in bacteria is transient, meaning that the expression of our tool in the bacteria is limited in time. Furthermore, rapamicyn should be added in the fermenter in order to fuse our tool, which could be an issue concerning the composition of the fermenter. This point is crucial and constitutes the go/no-go step of the commercialization of our project. A solution to this problem is described in the text part.

Production of non GMO stains for the industry and research

In the European context, associate a product under the title of GMO is not insignificant, because it is a synonym for interdiction. Thus the questioning on CRISPR/Cas9 technology is shifted directly from their evaluation to their potential interdiction through a classification as GMO.

As it was mentioned before, the wide majority of the strains used in the bio-industry are GMOs. To learn more about GMO regulation, click here.

There is a debate in the European Union in order to decide if organisms modified by CRISPR/Cas9 technology should be considered as GMO or not. Nevertheless, this debate is only for CRISPR/Cas9 doing a double strand break (DSB) in the chromosome and in the case of our tool, the Cas9 does not have an exonucleasic (meaning cutting) activity because it is composed of dCas9.

Furthermore, even if our system is used to modify an organism, it will be still considered as a genetic modification as we introduce foreign DNA (the expression plasmid) in the bacteria. But recent works on the use of CRISPR/Cas9 system help us to overcome this difficulty. Instead of transforming bacteria with the expression plasmid, it could be possible to introduce a dCas9 ribonucleoproteins (RNP), which consist of purified Cas9 protein in complex with a sgRNA.  They are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques (McDade, 2016). So it will be possible to deliver directly our tool by this method [Fig. 2c]. As no foreign DNA is introduced, the organisms edited by our tool should not be considered as GMOs!

To sum up

SCHEME

References

http://www.college-de-france.fr/site/en-cirb/espeli.htm

Lagomarsino MC, Espéli O, Junier I. From structure to function of bacterial chromosomes: Evolutionary perspectives and ideas for new experiments. FEBS Letters. 7 oct 2015;589(20PartA):2996‑3004. http://dx.doi.org/10.1016/j.febslet.2015.07.002

Li J, Zhang Y. Relationship between promoter sequence and its strength in gene expression. Eur Phys J E Soft Matter. sept 2014;37(9):44. http://link.springer.com/article/10.1140%2Fepje%2Fi2014-14086-1

Kim H, Kim J-S. A guide to genome engineering with programmable nucleases. Nature Reviews Genetics. 2 avr 2014;15(5):321‑34. https://www.ncbi.nlm.nih.gov/pubmed/24690881

Smith-Willis H, San Martin B. Revolutionizing genome editing with CRISPR/Cas9: patent battles and human embryos. Cell Gene Therapy Insights. 2015;1(2):253‑62. http://insights.bio/cell-and-gene-therapy-insights/?bio_journals=revolutionizing-genome-editing-with-crisprcas9-patent-battles-and-human-embryos

http://www.clontech.com/US/Products/Inducible_Systems/Protein-Protein_Interactions/iDimerize_Product_Overview

Gallage NJ, Møller BL. Vanillin–Bioconversion and Bioengineering of the Most Popular Plant Flavor and Its De Novo Biosynthesis in the Vanilla Orchid. Molecular Plant. janv 2015;8(1):40‑57. https://www.ncbi.nlm.nih.gov/pubmed/25578271

http://blog.addgene.org/genome-engineering-using-cas9/grna-ribonucleoproteins-rnps