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. 2015 Oct 10;43(18):8913–8923. doi: 10.1093/nar/gkv882

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© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Conjugation assays reveal DNA interference activity of the S. putrefaciens CN-32 CRISPR–Cas system. (A) The experimental set-up of the conjugation assay is depicted. Two plasmids were used: (I) a control plasmid (pNT, plasmid non-target) and (II) a plasmid containing a PAM sequence (light grey) and a sequence matching spacer 13 (dark grey) of the S. putrefaciens CN-32 CRISPR array (pT, plasmid target). The number of S. putrefaciens CN-32 colonies carrying each plasmid was counted and the conjugation efficiency was determined. Interference is observed if pT/pNT is lower than 1 in triplicate assays. (B) The pT/pNT ratio is calculated for conjugation assays into S. putrefaciens CN-32 wild type cells and strains containing deletions of genes encoding Cas1, Cas3, Cas1821, Cas1822 and H-NS. Additionally, a Cas3 active site variant (HDmut, H156A/D157A) was tested. (C) Northern blot analyses were performed with extracted total RNA from S. putrefaciens CN-32 wild-type and cas gene-knockout strains using a 5′-γ-[³²P]-ATP labeled probe complementary to the repeat sequence. A stable crRNA pool was absent in the S. putrefaciens CN-32 Δcas1821 and Δcas1822 strains.