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Period: 1993 BCE to
Discovery of CRISPR and its function
Francisco Mojica was the first to characterize a CRISPR locus in 1993, realizing that disparate repeat sequences shared common features later known as CRISPR hallmarks. He coined the term CRISPR in correspondence with Ruud Jansen, who published it in print in 2002. Mojica's 2005 report found CRISPR sequences matching bacteriophage genomes, leading him to hypothesize its adaptive immune system function. -
Discovery of Cas9 and PAM
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. -
Hypothetical scheme of adaptive immunity
"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
Danisco scientists sought to understand S. thermophilus’ response to phage attack, a common issue in industrial yogurt production. Horvath and colleagues experimentally proved CRISPR systems adapt like an adaptive immune system, integrating new phage DNA into the CRISPR array for defense. They also disclosed the role of Cas9 as the only protein needed for interference, thereby resolving unknown aspects of CRISPR system's operation. -
CRISPR acts on DNA targets
Marraffini and Sontheimer's 2008 paper demonstrated that CRISPR targets DNA, contradicting the widely accepted belief that it functions like eukaryotic RNAi, which targets RNA. Marraffini and Sontheimer also indicated in the paper that this system's potential extends beyond bacterial systems. (It should be noted, however, that another type of CRISPR system can target RNA (Hale et al., 2009)). -
Spacer sequences are transcribed into guide RNAs
"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)." -
Cas9 cleaves target DNA
"Moineau and colleagues demonstrated that CRISPR-Cas9 creates double-stranded breaks in target DNA at precise positions, 3 nucleotides upstream of the PAM (Garneau et al., 2010). They also confirmed that Cas9 is the only protein required for cleavage in the CRISPR-Cas9 system. This is a distinguishing feature of Type II CRISPR systems, in which interference is mediated by a single large protein (here Cas9) in conjunction with crRNAs." -
CRISPR systems can function heterologously in other species
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Discovery of tracrRNA for Cas9 system
The pivotal discovery in the natural CRISPR-Cas9-guided interference mechanism came from Emmanuelle Charpentier's group. Conducting small RNA sequencing on Streptococcus pyogenes, equipped with a Cas9-containing CRISPR-Cas system, they identified a second small RNA named trans-activating CRISPR RNA (tracrRNA) alongside the known crRNA. -
Biochemical characterization of Cas9-mediated cleavage
Virginijus Siksnys, Vilnius University, Lithuania
June, 2012 — Charpentier and Jennifer Doudna, University of California, Berkeley -
CRISPR-Cas9 harnessed for genome editing
Feng Zhang, Broad Institute of MIT and Harvard, McGovern Institute for Brain Research at MIT, Massachusetts