Perspective
In this project we design a tool for study the relation of DNA structure and gene expression. Here in perspective we propose many applications of our tool in genome study and industry. Using dCas9 in this system give us this advantage to target the desired sequence specifically and change structure of DNA. Indeed, it would be possible to design specific sgRNA to target specific sequences.
Study the chromosome structure and function
Our tool considers as a reporter for chromosome structural properties. According to the Lagomarsino et al. paper developing a stable reporter is the most important challenge in chromosome structural studies. (Lagomarsino et al., 2015).
One of the classic technics to study the contact between two arbitrary genomic loci is protein-fragment complementation assay (PCA). In 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. It is assumed that this assay may be naturally transposed to the problem of the contact between two genomic loci. Lagomarsino suggested in his paper that in order to end this problem, instead of considering two target proteins, they 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]. In our project we chose tripartite split-GFP as a marker.
A possible PCA-Cas9 assay to select bacteria on the basis of physical contacts between pairs of genomic loci. On the one hand, two short guide RNAs (sgRNA1 and sgRNA2) are used to recognize two specific genomic loci (in red and blue) distantly situated along the genome. On the other hand, the Cas9 protein, which is modified to have no endonuclease activity, is used in two different fused systems. Each fused system contains one moiety (X or Y) of a reporter protein (X–Y) that can confer either survival to the bacteria, as in a DHFR system (Remy et al. 2007) , or fluorescence, as in a FRET system (Miyawaki et al. 2011) . Both cases enable the selection of bacteria that have brought X and Y (i.e., the two genomic loci) into contact. See (Mali et al. 2013) for a more general discussion about the engineering potential of CRISPR-Cas9 systems.
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. With our tool we are able to study how we can change the strength of one desired promotor according to DNA topology. 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!
We believe that with our tool it is possible to increase or decrease the strength of a given promotor by changing the DNA topology. This method does not necessitate the insertion of new promotor on the bacterial genome. In this method it is enough to design two sgRNAs for 2 regions of DNA which are situated in the optimal distance to the target promotors. The online bioinformatics tools also are available to in order to design the sgRNA by bio-informatics (Kim & Kim, 2014) . When the plasmids coding for our tools (customized with designed sgRNAs) transformed to the bacteria, it is able to change the strength of a given promoter [Fig. 2b]. We suggested applications and advantages of our tool which are listed and described as follow.
For research
Our tool remains available on the iGEM Registry for other iGEM teams who want to use this method.
For the industry
But our tool seems also very useful for the industry. Indeed, during out meeting with X from Mondelez International, it appeared that some industrials were interested in our tool. Even if it won’t be possible to commercialize our tool, we thought about several questions that could prevent us to commercialize our tool.
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 it 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 strains 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 than compounds produced by chemical ways. Furthermore, the strains used are produced 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 next part.
Production of non GMO strains 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. Furthermore, the European populations are very sceptical on GMOs and products which could contained GMOs: in the case of flavors, there is a debate on the term of Natural Flavors for flavors obtained by genetic engineering.
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 consists 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
(A) Classic method: insert a plasmid to clone a strong promoter in front of the gene
(B) iJ’Aime method 1: insert a plasmid to express iJ’AIME tool
(C) iJ’Aime method 2: insert iJ’AIME ribonucleoprotein
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
P. Mali , K.M. Esvelt , G.M. Church , Cas9 as a versatile tool for engineering biology. Nat. Methods, 10, 10 (2013), 957– 963.
I. Remy , F.X. Campbell-Valois , S.W. Michnick , Detection of protein–protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc., 2, 9 (2007), 2120– 2125.
A. Miyawaki , Development of probes for cellular functions using fluorescent proteins and fluorescence resonance energy transfer. Annu. Rev. Biochem., 80, (2011), 357– 373.
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
