The New Microbiology. Pascale Cossart

bacteria they target.

      To begin their attack, bacteriophages need a site of attachment, a particular component on the surface of a bacterium. This site of attachment is specific for each virus and the bacteria that it can infect.

      Infections of bacteria by phages are of great concern, particularly in the dairy industry, which uses certain bacteria, for example Streptococcus thermophilus, to make yogurt and cheese. S. thermophilus transforms the lactose in milk into lactic acid. Additionally, each different bacterial strain contributes its own unique taste and texture to the yogurt, which must remain consistent to ensure a reliable product and successful sales. If a bacterial strain disappears as the result of a bacteriophage infection, the economic consequences for the manufacturer can be disastrous.

Figure 9. Bacteriophages infecting an Escherichia coli bacterium.

      One of the great discoveries of this decade is that bacteria have an immune system called CRISPR, for clustered regularly interspaced short palindromic repeats, meaning small, regularly spaced, palindromic (the sequence reads the same from either end) repeats. CRISPR regions in the chromosomes allow bacteria to recognize predators, particularly previously encountered phages, and to destroy them. CRISPR regions protect and essentially “vaccinate” bacteria against bacteriophages.

      In fact, it has been shown that bacteria can be artificially vaccinated! When a population of bacteria is inoculated with a phage, a small number survive and are able to integrate a fragment of the phage DNA into their genome, in the region called the CRISPR locus. This allows the bacteria, if the phage ever attacks again, to recognize the phage DNA and degrade it. This ingenious phenomenon, known as interference, occurs due to the structure of the CRISPR region and to cas genes (CRISPR-associated genes) located near this region.

The CRISPR locus is a region of the chromosome composed of repeated sequences of around 50 nucleotides, interspersed with sequences known as spacers that are similar to those of bacteriophages. Some bacteria have several CRISPR loci with different sequence repetitions. Around 40% of bacteria have one or more CRISPRs, whereas others have none. CRISPR loci can be quite long, sometimes with more than 100 repetitions and spacers. CRISPRs have two functions: acquisition and interference. Acquisition, also called adaptation, is the process of acquiring fragments of DNA from a phage, and interference is the immunization process by Cas proteins encoded by cas genes (Fig. 10).

      Bacteria have numerous proteins with various complementary and synergistic functions in the process of adaptation and interference. They permit the addition of DNA fragments into the CRISPR locus, but their main purpose is to react to invading phages. The CRISPR locus is transcribed into a long CRISPR RNA, which is then split into small RNAs called crRNAs, each containing a spacer and a part of the repeated sequence. When a phage injects its DNA into the bacterium, the crRNA recognizes and binds to it. An enzyme then recognizes the hybrid and cleaves the phage DNA at the point where the crRNA has paired. Replication of the phage DNA is inactivated, and the infection is stopped.

      One of the key discoveries that led to the use of CRISPR systems in what is called “genome editing,” or modification, was the identification of the proteins involved in the cleavage of the hybrid DNA. This process is performed by a complex of proteins containing the protein Cas1 and sometimes by a single protein called Cas9. Cas9 is unique in that it can attach itself to a DNA strand and, due to the two distinct domains of its structure, cut this DNA on each of its two strands. This protein is the basis of the CRISPR/Cas9 technology, which enables a variety of genome modifications and mutations in mammals, plants, insects, and fish in addition to bacteria. This system works due to the Cas9 protein and also a guide RNA hybrid that is made from one RNA similar to the region to be mutated and a second RNA called tracrRNA, or trans-activating crRNA. tracrRNA was discovered next to the CRISPR locus in Streptococcus pyogenes and was shown to be homologous to the repeated regions of the locus, enabling it to guide the Cas9 protein and the crRNA toward the target.

      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.