CRISPR/Cas9 is a genome editing tool based on the protein Cas9, which acts as a pair of molecular scissors that can cut portions of target DNA and can be specifically programmed to alter a very precise point in the genome of animal, human or plant cells.
When Cas9 cuts into the genome, scientists can remove sequences of DNA that are damaged, or replace these sequences with healthy ones, thus for example eliminating the mutations that cause serious diseases.
Cas9 is programmed with an RNA molecule, called guide RNA, which directs it to a specific target. This molecule can be easily modified in laboratory and, once it is combined with Cas9, forms a sort of leash for Cas9, binding it only to the chosen target sequence.
Originally, CRISPR/Cas9 was developed from studies on bacteria, where the Cas9 protein works as a molecular scissors helping these microorganisms protect themselves from pathogenic viruses, acting like the bacteria’s immune system.
Between 2012 and 2013 two US research teams (from Berkley University and the MIT in Boston) were the first to prove that this bacteria-inspired technology could be used in biotechnology to cut specific sequences of DNA in the genome of a non-bacterial cell.
This discovery was a turning point biomedical research because, for the first time, scientists were able to alter human genome in a simple, effective, fast and economic way. That is why CRISPR/Cas9 spread to laboratories all over the world in very little time and it is used today both in basic research and for practical applications. In fact, despite being a rather new and rapidly evolving technology, it is so powerful that it will soon be used in clinical trials.
For the first time, with CRISPR/Cas9 we can start to think that it is possible to treat a wide range of genetic disorders for which there was no therapeutic approach.
Towards a high-precision genome editor
The huge potential of the genomic scissors that is CRISPR/Cas9, the genome editor, in a therapeutic setting, has been hampered so far by off-target cuts, that are cuts in unintended sites in the human genome.
Researchers at CIBIO started long ago to work on how to make this technology more precise and suitable to be used in clinical settings, when it was still emerging and it had not shown all of its potentialities. They identified a safe and efficient method to transfer CRISPR/Cas9 to the cells to be treated (lentiSLiCES), which has been the subject of an article published in Nature Communications, and devised a new technique to identify more precise CRISPR/Cas9 variants, like CRISPR/evoCas9, as described in a paper published in Nature Biotechnology.
Now CIBIO’s researchers aim to develop other further techniques for improved genome editing.
The project of CIBIO can be summed up in these 4 experimental objectives.
(1) Optimizing the transfer of the genome editor
Genome editing tools, including the high-precision evoCas9 molecule, must be efficiently transferred into the “damaged” cells whose DNA is altered.
At the moment, there are two reasons that block this transfer: the size of the editing tools, which are too big to be compatible with the vectors used in gene therapy, and biological barriers, including the membranes that surround cells.
Using the screening method in yeast adopted to develop evoCas9, CIBIO aims to develop smaller genome editing tools that can be easily transferred.
To reach their goal, researchers will include in the screening many Cas9 variants that have recently been discovered by different research teams around the world which, however, are not yet working in human cells.
(2) Strengthening gene removal techniques
The technology of CRISPR/Cas9 led to significant developments in the techniques through which sequences of mutated genes are replaced with healthy ones.
With this technology it is possible to replace single genes or larger portions of DNA showing multiple mutations.
However, genome editing through CRISPR/Cas9 as gene replacement method is still far from clinical practice because it is still largely inefficient.
Researchers at CIBIO aim to carry out studies to identify cell factors capable of facilitating the molecular mechanisms that enable gene replacement within the cell when they can be replaced with healthy sequences, to stimulate their functioning.
(3) Evolution of mutant proteins for their engineering in genetic disorders
In many genetic disorders proteins do not work properly.
The goal of genome editing is to correct these malfunctions and restore the normal cell functioning processes.
However, some mutations are very difficult to eliminate because of the position of genes in the DNA or because of the technical limitations of the genome editing tool.
Research at CIBIO will work to find a solution to these problems through an approach that aims to evolve the mutated protein instead of correcting the mutation.
In this framework CRISPR/evoCas9, the high-precision genome editor, will be used to edit the mutated gene in a way to eliminate the genetic damage that keeps the affected gene from functioning.
(4) Compensating genetic alterations to increase protein function
The CRISPR/evoCas9 genome editor cannot be successfully employed in the case of many genetic disorders which are caused by an altered or non-functioning gene because of a gene mutation.
The majority of genetic disorders are recessive, therefore called recessive genetic disorders.
Researchers at CIBIO aim to modify the CRISPR/evoCas9 genome editor so that it can increase the gene function and make up for this faulty functioning.