¿Qué es el CRISPR?

Discovery of CRISPR
The story starts in the Mediterranean port of Santa Pola on Spain’s Costa Blanca, where the beautiful coast and vast salt marshes have for centuries attracted vacationers, flamingoes, and commercial salt producers. (The geography of the story is shown in Figure 2.) Francisco Mojica, who grew up nearby, frequented those beaches, and it was no surprise that, when he began his doctoral studies in 1989 at the University of Alicante, just up the coast, he joined a laboratory working on Haloferax mediterranei, an archaeal microbe with extreme salt tolerance that had been isolated from Santa Pola’s marshes. His advisor had found that the salt concentration of the growth medium appeared to affect the way in which restriction enzymes cut the microbe’s genome, and Mojica set out to characterize the altered fragments. In the first DNA fragment he examined, Mojica found a curious structure—multiple copies of a near-perfect, roughly palindromic, repeated sequence of 30 bases, separated by spacers of roughly 36 bases—that did not resemble any family of repeats known in microbes (Mojica et al., 1993).

The 28-year-old graduate student was captivated and devoted the next decade of his career to unraveling the mystery. He soon discovered similar repeats in the closely related H. volcanii, as well as in more distant halophilic archaea. Combing through the scientific literature, he also spotted a connection with eubacteria: a paper by a Japanese group (Ishino et al., 1987) had mentioned a repeat sequence in Escherichia coli that had a similar structure, although no sequence similarity, to the Haloferax repeats. These authors had made little of the observation, but Mojica realized that the presence of such similar structures in such distant microbes must signal an important function in prokaryotes. He wrote up a paper reporting this new class of repeats (Mojica et al., 1995) before heading off for a short post-doctoral stint at Oxford.

Mojica returned home to take up a faculty position at the University of Alicante. Because the school had hardly any start-up funds or lab space, he turned to bioinformatics to investigate the strange repeats, which he dubbed short regularly spaced repeats (SRSRs); the name would later be changed, at his suggestion, to clustered regularly interspaced palindromic repeats (CRISPR) (Jansen et al., 2002, Mojica and Garrett, 2012).

By 2000, Mojica had found CRISPR loci in 20 different microbes—including Mycobacterium tuberculosis, Clostridium difficile, and the plague bacteria Yersinia pestis (Mojica et al., 2000). Within 2 years, researchers had doubled the census and cataloged key features of loci—including the presence of specific CRISPR-associated (cas) genes in the immediate vicinity, which were presumably related to their function (Jansen et al., 2002). (Table 1 summarizes the modern classification of CRISPR systems.)

But what was the function of the CRISPR system? Hypotheses abounded: it was variously proposed to be involved in gene regulation, replicon partitioning, DNA repair, and other roles (Mojica and Garrett, 2012). But most of these guesses rested on little or no evidence, and one by one they proved to be wrong. As with the discovery of CRISPR, the critical insight came from bioinformatics.

CRISPR Is an Adaptive Immune System
During the August holiday in 2003, Mojica escaped the scorching heat of Santa Pola’s beaches and took refuge in his air-conditioned office in Alicante. By now the clear leader in the nascent CRISPR field, he had turned his focus from the repeats themselves to the spacers that separated them. Using his word processor, Mojica painstakingly extracted each spacer and inserted it into the BLAST program to search for similarity with any other known DNA sequence. He had tried this exercise before without success, but the DNA sequence databases were continually expanding and this time he struck gold. In a CRISPR locus that he had recently sequenced from an E. coli strain, one of the spacers matched the sequence of a P1 phage that infected many E. coli strains. However, the particular strain carrying the spacer was known to be resistant to P1 infection. By the end of the week, he had slogged through 4,500 spacers. Of 88 spacers with similarity to known sequences, two-thirds matched viruses or conjugative plasmids related to the microbe carrying the spacer. Mojica realized that CRISPR loci must encode the instructions for an adaptive immune system that protected microbes against specific infections.

Mojica went out to celebrate with colleagues over cognac and returned the next morning to draft a paper. So began an 18-month odyssey of frustration. Recognizing the importance of the discovery, Mojica sent the paper to Nature. In November 2003, the journal rejected the paper without seeking external review; inexplicably, the editor claimed the key idea was already known. In January 2004, the Proceedings of the National Academy of Sciences decided that the paper lacked sufficient “novelty and importance” to justify sending it out to review. Molecular Microbiology and Nucleic Acid Research rejected the paper in turn. By now desperate and afraid of being scooped, Mojica sent the paper to Journal of Molecular Evolution. After 12 more months of review and revision, the paper reporting CRISPR’s likely function finally appeared on February 1, 2005 (Mojica et al., 2005).