5 research outputs found

    PAM and repeat-end correlation.

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    <p>(A): PAMs of observed spacers and the co-occurring trinucleotide repeat-ends associated with these spacers. Notice that the spacer-proximal nucleotide of the repeat end is identical to the protospacer-proximal nucleotide of the PAM. (B): Schematic of the proposed mechanism for spacer acquisition during CRISPR adaptation. A protospacer with specific PAM is selected after which it is processed into the pre-spacer (at least 33–34 bp), which contains the last nucleotide of the PAM (the pre-spacer could be single-stranded or double-stranded). The pre-spacer is than integrated at the leader proximal end of the CRISPR locus. The nucleotide derived from the PAM forms the last nucleotide of the repeat. (C): R-loop formation by mature crRNA (61 nucleotides) during CRISPR interference. Notice that the last nucleotide of the repeat (the nucleotide derived from the PAM) is complementary to the target DNA sequence. It remains unknown whether base-pairing between these nucleotides is important for interference.</p

    Linear display of pRSF-1b and locations of protospacers.

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    <p>The (+) and (−) strands and corresponding protospacers are coloured red and black, respectively. Kanamycin marker (Kan), Origin of replication (Ori) and <i>lacI</i> (LacI) are shown as arrows. Protospacers have an AAG PAM unless indicated otherwise.</p

    Model of the strand specific positive feedback loop.

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    <p>Cells with a spacer against a known and actively present invader DNA produce targeting Cascade complexes in the expression stage. In the interference stage, Cascade binds the target dsDNA after which the target is cleaved and degraded by Cas3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888-Westra1" target="_blank">[15]</a>. DNA degradation products generated by Cascade and Cas3 (which could be ssDNA or dsDNA) act as precursors for new spacers in the adaptation phase in a strand-specific manner. By integration of these strand-specific precursors, the spacer repertoire against an actively present invader is expanded, completing the positive feedback loop.</p

    Effect of integrated spacers on retransformation efficiency.

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    <p>Transformation efficiencies of various PIMs and the control (Wild type <i>E. coli</i> K12 W3110) are given in a logarithmic scale as colony forming units (CFU) per µg of pRSF-1b plasmid DNA. For each PIM, the number of spacers integrated in either CRISPR locus 2.1 or 2.3 is given. All spacers have an AAG PAM, unless indicated otherwise. The exact spacer composition of each PIM is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>.</p

    Graphical representation of AG and GC contents of each observed and possible spacer.

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    <p>Observed spacers (⧫) are spacers integrated in CRISPR loci 2.1 and 2.3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035888#pone.0035888.s001" target="_blank">Table S1</a>). These spacers are 32 or 33-mers with various PAMs. Possible spacers (Ο) are all 32-mers found on pRSF-1b directly downstream of an AAG PAM.</p