Discovery of CRISPR and its function 1993 - 2005 — Francisco Mojica, University of Alicante, Spain Francisco Mojica was the first researcher to characterize what is now called a CRISPR locus, reported in 1993. He worked on them throughout the 1990s, and in 2000, he recognized that what had been reported as disparate repeat sequences actually shared a common set of features, now known to be hallmarks of CRISPR sequences (he coined the term CRISPR through correspondence with Ruud Jansen, who first used the term in print in 2002). In 2005 he reported that these sequences matched snippets from the genomes of bacteriophage (Mojica et al., 2005). This finding led him to hypothesize, correctly, that CRISPR is an adaptive immune system. Another group, working independently, published similar findings around this same time (Pourcel et al., 2005) Discovery of Cas9 and PAM May, 2005 — Alexander Bolotin, French National Institute for Agricultural Research (INRA) Bolotin was studying the bacteria Streptococcus thermophilus, which had just been sequenced, revealing an unusual CRISPR locus (Bolotin et al., 2005). Although the CRISPR array was similar to previously reported systems, it lacked some of the known cas genes and instead contained novel cas genes, including one encoding a large protein they predicted to have nuclease activity, which is now known as Cas9. Furthermore, they noted that the spacers, which have homology to viral genes, all share a common sequence at one end. ![]() This sequence, the protospacer adjacent motif (PAM), is required for target recognition. Hypothetical scheme of adaptive immunity March, 2006 — Eugene Koonin, US National Center for Biotechnology Information, NIH Koonin was studying clusters of orthologous groups of proteins by computational analysis and proposed a hypothetical scheme for CRISPR cascades as bacterial immune system based on inserts homologous to phage DNA in the natural spacer array, abandoning previous hypothesis that the Cas proteins might comprise a novel DNA repair system. (Makarova et al., 2006) Experimental demonstration of adaptive immunity March, 2007 — Philippe Horvath, Danisco France SAS S. Thermophilus is widely used in the dairy industry to make yogurt and cheese, and scientists at Danisco wanted to explore how it responds to phage attack, a common problem in industrial yogurt making. Horvath and colleagues showed experimentally that CRISPR systems are indeed an adaptive immune system: they integrate new phage DNA into the CRISPR array, which allows them to fight off the next wave of attacking phage (Barrangou et al., 2007). Furthermore, they showed that Cas9 is likely the only protein required for interference, the process by which the CRISPR system inactivates invading phage, details of which were not yet known. ![]() AlfaSelect Air is our state-of-the-art configuration and selection software for Alfa Laval air heat exchangers. Cropes hnl patch za pes6 demon. AlfaSelect Air facilitates configuration & selection and make our customers stay in control during all stages of this crucial process. Alfa Laval – a world leader in heat transfer, centrifugal separation and fluid handling – has signed an umbrella agreement with the LU-VE Group to sell parts of its air heat exchanger business. Spacer sequences are transcribed into guide RNAs August, 2008 — John van der Oost, University of Wageningen, Netherlands Scientists soon began to fill in some of the details on exactly how CRISPR-Cas systems “interfere” with invading phage. The first piece of critical information came from John van der Oost and colleagues who showed that in E-scherichia coli, spacer sequences, which are derived from phage, are transcribed into small RNAs, termed CRISPR RNAs (crRNAs), that guide Cas proteins to the target DNA (Brouns et al., 2008). CRISPR acts on DNA targets December, 2008 — Luciano Marraffini and Erik Sontheimer, Northwestern University, Illinois The next key piece in understanding the mechanism of interference came from Marraffini and Sontheimer, who elegantly demonstrated that the target molecule is DNA, not RNA (Marraffini and Sontheimer, 2008). This was somewhat surprising, as many people had considered CRISPR to be a parallel to eukaryotic RNAi silencing mechanisms, which target RNA. Marraffini and Sontheimer explicitly noted in their paper that this system could be a powerful tool if it could be transferred to non-bacterial systems. (It should be noted, however, that a different type of CRISPR system can target RNA (Hale et al., 2009)).
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