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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 4. — Analysis of the DNA target specificity of the S. putrefaciens CN-32 CRISPR–Cas system. Conjugation assays (see Figure 3) were used to analyze plasmid DNA target variants for DNA interference in S. putrefaciens CN-32 cells. (A) Interference was observed if the transferred DNA strand contained a sequence complementary to the crRNA13 (sp13-GG) or a sequence identical with spacer13 (sp13-GG*). Interference was evident for the wild-type and ΔH-NS strains, which contained all cas genes. However, the deletion of cas3 (Sputcn32_1820) or cas1822 abolished interference activity. The presence of the PAM sequence GG at the 3′ end of the DNA target (5′-protospacer-PAM-3′) was essential for the interference activity and PAM mutants (sp13-AG, sp13-GA, sp13-AA or sp13-CC) and a 10 nt poly-A sequence disrupting crRNA/DNA complementarity (sp13-polyA) were not targeted. (B) DNA interference activity was dependent on spacer sequence or crRNA abundance (spacers 1,3,4,13,15,20,34, see Figure 2).