Review: CRISPR-CAS9, Genetic Editing Technology

Review: CRISPR-CAS9, Genetic Editing Technology 

Yahia BELLOUCHE

The explosive development in the domains of microscopy, data analysis and molecular biology have led us to a better understanding of cell functioning. Basic the sequence gene-protein- function might be, further mapping of the different loci and their products allowed a larger view on the pathophysiology of many illnesses, that said, almost every pathological process we could meet in the different entities, contain a quantitative and/or qualitative disorder in the sequence above. All that opened the debate on the importance of the genome and the potential benefits of the technologies of genetic editing such as the CRISPR-CAS9 system, subject of our discussion.


Basic concepts 

Every Cell in the body holds a complete copy of the genome, which is a set of DNA chains, organized in different structural levels, resulting from chemical links between millions of nucleic acids. Each gene occupies a definite place called a locus that codes for at least one “functional molecule”, proteins mostly, that induce a given change somewhere in the cell or out of intracellular compartment. 

The sequence explained above is not in fact simple, many processes follow up to the final effector. Copies are made of the gene in the form of mRNA after transcription, they transit into the cytosol and get translated by means of polysomes onto proteins that can submit some modification (glycosylation, cleavage, conformational maturity….), before they get addressed to the corresponding compartment where they accomplish the tasks supposed. This model implies an “On demand” gene expression system rather than a “Continuous” one. The general regulation loop scheme is very much valid In this context, this means that a stimulus can induce which gene to be expressed and when, recent data suggests further control of external milieu, one that commands how and what is transcribed in a gene, which means that a phenotype is in fact, an interaction product between an intrinsic genetic code and some surrounding conditions, study subject for Epigenetics. As a result, the environment of a cell and its level of differentiation define various functional stages where a cell performs different tasks and exposes many changes. The pool of proteins expressed by a cell in a given functional state is known as a proteome, its field of study Proteomics. Same definitions can be applied to the genome, genomics respectively. 

Considering now the pathological side, an interactive and dynamic genome, beneficial for a cell on one hand, would only mean extra vulnerability to modifications and alterations on the other. A fact that can be largely deduced from the genetic analysis of affected cells from many examples such as cancer, viral infections and inflammatory response. However, those are extremely rare, and even with the presence of many potential alteration inducers, the genome is somewhat sane, this has been studied largely through the last decade and many protection systems were discovered, the CRISPR-CAS9 being one of those.


CRISPR-CAS9 system in prokaryotes 

Experimental data from the 1980s discovered that species of E.Coli could resist a viral infection after a few generations, raising a question about the mechanisms. Bacteriophages (viruses infecting bacteria) contain some genetic sequences that –after penetration- get integrated in the bacteria genome causing alterations and modifications through transcription. These sequences code for viral structure proteins and mandatory enzymatic material. Throughout the natural history of infection, some colonies begin to expose a certain degree of resistance, documented by the deceleration of viral multiplication rates. Further analysis of the bacterial genome detected some DNA sequences originating from the virus itself. The questions about the mechanisms were not solved until 2007 with the work of Barrangou & Col, where the CRISPR-CAS system was well understood. Studies have concluded that some gene loci in the genome were the association result of pieces of repetitive sequences and some viral sequences called spacers. 

That said, the CRISPR loci transcription product was found to be a special type of RNA (called crRNA) that had the ability to bind a set of proteins such as CAS, the resistance to viral infection appears after two phases, important each, to understand the mechanisms underlying this genetic protection gate against external aggressions.

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 cytosol captured from the penetration of a particle. Specialized proteins such as Cas 1 and Cas 2 are involved in the capture, processing, and integration of the spacers 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 transcription scheme in the rest of the process, the CRISPR 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 sequence crRNA is obtained, guaranteeing a high-level recognition ability towards any viral products generated 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 standard nucleic acids binding characteristics (basic complementarity), that means that the crRNA work almost like “tags” or markers that underlie where the endonucleases need to cleave, assuring the destruction of the viral DNA and protection of the cell from the results of their intracellular multiplication, important to remind that this mechanism is highly specific, and very similar to the multicellular adaptive immunity as of the dynamic and the conditions of activation.

CRISPR – CAS9 as an editing tool

Area of Interest 

After these data about the function and the characteristics of the CRISPR-CAS9 system, the spectrum of potential 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 sequences and incorporate them as spacers, and instead of lysis, we would be able through molecular engineering of the enzymatic part of the complex, to add, delete 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 revolutionize therapies as of its highly accurate, robust and fundamental effect on the cell function, the genetic editing, 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 technology 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 characteristics and decrease immunisation risks. All these procedures are based on a vehicle (Plasmid usually) that carries the operon to the bacterial genome, enhanced with promotors and localization sequences.

2. Genetic editing and cancer 

The development of genomics has allowed a more analytic approach as of the variations of genes expression, and the neo-mutation through the natural selection of the dominant clone of a tumour, with a whole group of reversibility tools, which may even in the later stages of a cancer, diminish its hostility, with the theoretical possibility of a highly selective, efficient and adverse effect-free cure for cancer. 

3. Cell aging and regeneration therapy 

The study of embryogenesis and organs morphogenesis provided a deeper understanding of the genetic regulation of these processes, which can open the doors wide towards the development of autogeneic in vitro transplants growing. Besides, the elevation of growth rates of some tissue can be a better mechanism to extinct the effect of some degenerative pathologies, with the general optic of mastering cell aging and regeneration technology.

DNA breaks and pathways of genetic editing

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 - Image d’arrière plan par starline / Freepik