Figure 1. Type II CRISPR-Cas systems(a) Genetic organization of the type II-A CRISPR-Cas system of Streptococcus pyogenes SF370. The cas operon is composed of four genes, cas9, cas1, cas2, and csn2; the last three are thought to be involved in the acquisition of new spacer sequences. This operon is followed by the CRISPR array containing seven repeats (white boxes, 36 nt long) and six spacers (numbered, colored boxes, 30 nt long) that match different S. pyogenes bacteriophages. Preceding the cas operon and transcribed in the other direction is the trans-activating CRISPR RNA (tracrRNA) gene, which encodes a small RNA with homology to the repeat sequences. (b) CRISPR RNA (crRNA) processing. The CRISPR locus is transcribed as a long precursor (the crRNA precursor) containing repeats (grey line) and spacers (colored lines). Assisted by Cas9, the tracrRNA interacts with each repeat sequence to generate a double-stranded RNA (dsRNA) that is cleaved by RNase III, thus liberating the small crRNAs from the precursor. Further processing at the 5′ end of the crRNA shortens the length of the guide sequence to 20 nt. Black arrowheads indicate the sites of RNA processing. (c) Targeting. Cas9 scans the genome (dsDNA) of the invader to find a region of complementarity with the guide crRNA and introduces a dsDNA cut using two independent nuclease domains, one for each DNA strand, RuvC and HNH. A requirement for cleavage is the presence of an NGG sequence (known as protospacer adjacent motif, or PAM) immediately downstream of the target site. Abbreviations: cas, CRISPR-associated; CRISPR, clustered regularly interspaced short palindromic repeats; nt, nucleotide.
Image published in: Bikard D and Marraffini LA (2013)
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