Pascale Cossart - The New Microbiology

In summary, by expressing the Cas9 protein with a composite RNA made up of an identical sequence to the target region, a tracrRNA, and a complementary fragment to the tracrRNA, one can now introduce a mutation or deletion into a target genome of any origin.

After the 2012 publication in Science of the elegant studies by the teams led by Emmanuelle Charpentier and Jennifer Doudna, the CRISPR method was so intriguing that it provoked an avalanche of research and publications demonstrating that this technique could be used in many cases and with many variations. For example, a Cas9 protein named dCas (dead Cas), if fused to a repressor or activator protein, can attach to the desired locus without cleaving it, then activate or repress genes in mammals. It was also shown that the CRISPR method can generate multiple mutations at a time if performed with a single Cas9 but with a variety of guide RNAs.

Figure 10. (Top) The three steps involved in CRISPR function. (1) Integration of a piece of DNA from a phage into the CRISPR locus (acquisition); (2) the expression of Cas proteins and of pre-crRNA, which is then split into small crRNAs; (3) the interference that takes place when the DNA injected by a phage into a bacterium meets a crRNA, a hybrid form that is then degraded, consequently preventing infection. (Bottom) Schematic drawing of genome modification (gene editing) by an sgRNA (small guided RNA) made of crRNA and tracrRNA and the endonuclease Cas9.

It is thus through the studies of microbiologists interested in fundamental aspects of bacterial physiology—such as resistance to phages, the role of the noncoding repeated sequences found in many genomes, and the role of small noncoding RNAs—that a revolutionary technique was born. CRISPR has revolutionized many domains of biology, to the point that medical applications for this technique are now within reach, such as targeted gene therapy. The researchers most involved in these discoveries have already been recognized for their contributions by undoubtedly well-deserved awards.

While being the object of intense study, the CRISPR/Cas9 technology raises important ethical questions. Should we start using it now for gene therapy? Do we have enough experience and perspective on the matter to make this decision? How can we be sure we are not creating unintentional mutations along with the targeted mutations? Should we have ethical concerns even with the latest technical developments and the use of modified Cas9 proteins? These issues are the focus of important international ethics committees.

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