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].
(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
Production of non-GMO strains for the industry
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. In our tool we use dCas9 proteins, the Cas9 protein in which the exonuclease (cutting) activity is enabled.
However, our system still can be considered as a genetic modification of bacteria (expression plasmid containing foreign DNA). But recent works on the use of CRISPR/Cas9 system help us to overcome this issue as well. It is possible to introduce the dCas9 ribonucleoproteins (RNP), which consist of purified dCas9 protein in complex with a sgRNA instead of transformation of coding plasmids. They are assembled in vitro and can be delivered directly to cells using standard electroporation or transfection techniques (McDade and Waxmonsky ,2016) .In conclusion, it is possible to deliver directly our tool by this method [Fig. 2c] . As no foreign DNA is introduced, the organisms manipulated by our tool should not be considered as GMOs!
References
