ReMed 2018 ReMed Magazine N°4 - Cutting Edge | Page 13

important each, to understand the mechanisms under- lying this genetic protection gate against external ag- gressions. their intracellular multiplication, important to remind that this mechanism is highly specific, and very similar to the multicellular adaptive immunity as of the dy- namic and the conditions of activation. Figure -1- Genomic adaptive immunity in prokaryotes (Horvath P, Barrangou R (January 2010). "CRISPR/Cas, the immune system of bacteria and archaea) 1. Acquisition phase In this phase, the bacteria acquires the viral sequences through the processing of the viral genome in the cy- tosol captured from the penetration of a particle. Spe- cialized proteins such as Cas 1 and Cas 2 are involved in the capture, processing, and integration of the spac- ers between the CRISPR loci arrays, which announces the implementation of genetic adaptive immunity against the virus, and the activation of the CRISPR loci activation. 2. Loci transcription and Biogenesis There isn’t much of an exception to standard DNA tran- scription scheme in the rest of the process, the CRIS- PR loci is transcribed to generate a pmRNA, this last, with the intervention of another set of CAS proteins is cleaved to form many pieces crRNA that can bind to the endonucleases necessary to guide their lysis function. Many enzymes are implicated in the process of viral genome cleavage, CAS9 being the most studied among them all. 3. Interference After the two steps, a complex of an endonuclease with a non-specific DNA cleavage function and a specific se- quence crRNA is obtained, guaranteeing a high-level recognition ability towards any viral products generat- ed inside the cell in the course of the viral cycle. This action requires the special binding between the crRNA and the viral DNA sequence, with respect to the stan- dard nucleic acids binding characteristics (basic com- plementarity), that means that the crRNA work almost like “tags” or markers that underlie where the endonu- cleases need to cleave, assuring the destruction of the viral DNA and protection of the cell from the results of Figure -2- CRISPR – CAS9 as an editing tool Area of Interest After these data about the function and the character- istics of the CRISPR-CAS9 system, the spectrum of po- tential applications appeared to be extremely large, for it provides a high level of specificity for any action it would be associated with. That means we can program any locus in any genetic material of any cell we want, provided that we acquire the corresponding DNA se- quences and incorporate them as spacers, and instead of lysis, we would be able through molecular engineer- ing of the enzymatic part of the complex, to add, de- lete and replace sequences which can lead to desired modifications on the cellular level, or correction of an alteration in a given pathology process. This can revo- lutionize therapies as of its highly accurate, robust and fundamental effect on the cell function, the genetic ed- iting, once technologically mastered, can provide many solutions and open paths towards understanding some of our era’s most complex pathologies Application examples As explained above, the possibilities of such a technol- ogy are unlimited with access to many domains. 1. Industrial genetic engineering The genetic editing of the recombinant DNA sequences can help raise the rates of expression and with it, the production capacity, improve pharmacological char- acteristics and decrease immunisation risks. All these procedures are based on a vehicle (Plasmid usually) that carries the operon to the bacterial genome, en- hanced with promotors and localization sequences. ReMed Magazine - Numéro 4 13