What history tells us CRISPR-Cas: The discovery of an immune system in prokaryotes

An increasing number of articles and reviews are being published that demonstrate the efficiency of the CRISPR-Cas9 system in genome editing of very different types of organisms and describe the resulting experimental and therapeutic perspectives. A canonical historical record of the major steps that led to the present situation has already been reported (see, for instance, Horvath and Barrangou 2010 and Doudna and Charpentier 2014). In 1987, a new type of repeated sequence was discovered in prokaryotes (Ishino et al. 1987). Between 2000 and 2002 the major characteristics of these short, regularly spaced repeats were described: they consist of clusters of short repeated palindromic sequences of 20-40 bp separated by unique intervening 20-60 bp sequences (Mojica et al. 2000). They are present in most archaea and a large percentage of bacteria. In 2002, Ruud Jansen and colleagues (Jansen et al. 2002) introduced a new term to designate these sequences – clustered regularly interspaced short palindromic repeats (CRISPRs), and showed that theywere often found close to a family of genes already described as a ‘DNA repair system’ (Makarova et al. 2002) and now called ‘Cas’ for ‘CRISPR-associated’. However, the origin of the spacer sequences remained unknown and it was only three years later that three groups independently showed that some of these spacer sequences have a bacteriophage origin (Mojica et al. 2005; Pourcel et al. 2005; Bolotin et al. 2005). In 2006, Eugene V Koonin and his collaborators suggested that the CRISPR-Cas system was a prokaryotic RNA interference–based immune system (Makarova et al. 2006). In 2007, Rodolphe Barrangou, Philippe Horvath and their collaborators (Barrangou et al. 2007) clearly demonstrated that the CRISPR-Cas system provides acquired immunity against bacteriophages: infection leads to the formation in a polarized way of new spacer sequences and to protection against further infection, which is abolished by the deletion of the spacer sequences or of some of the Cas genes. In the following years, the molecular mechanisms were partially unveiled and the structural and functional characteristics of the proteins encoded by theCas genes described, opening theway to the design of the CRISPR-Cas9 editing system. In this article, I will focus on the early steps and particularly on the multiplicity of explanations that were initially provided for the existence of the CRISPR-Cas system. I will devote the major part of this contribution to the three 2005 publications. In contrast with the previous historical description, these three articles did not simply demonstrate the bacteriophage origin of some of the spacer sequences, but they collectively reached the conclusion that the CRISPRCas system was a memory of infection and a defence system against further infections. They provided information on the mechanism of insertion of the new spacers, and proposed hypotheses on the way these spacers prevented further infection. Therefore, the issue is what the 2006 and 2007 publications really provided and why the 2005 contributions were not better recognized?