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. 2016 Apr 16;44(9):4266–4277. doi: 10.1093/nar/gkw260

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© The Author(s) 2016. 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-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 3. — Sequences spanning the leader-repeat junction are important for adaptation. (A) Sequences spanning the leader-repeat junction are conserved among haloarchaeal CRISPRs with similar repeats. Note that, for each CRISPR, the first repeat and its upstream ∼110-bp sequence were together retrieved and aligned (see Supplementary Figure S2 for details), and only a part of this multi-alignment is shown here. Using the WebLogo server (http://weblogo.berkeley.edu/logo.cgi), a sequence logo was generated from this multi-alignment. The inverted repeats (IR1 and IR2) within the CRISPR repeat are indicated. The full circle (•) indicates the position where an extra nucleotide from the Hlac132P repeat resulted in a gap in all the other repeat sequences during alignment. (B) Illustration of a series of a-CRISPR constructs with mutations (in blue) preceding, following, or flanking the leader-repeat junction. The repeat sequence is shown in brackets. Sequences between vertical arrows were duplicated during spacer integration. (C) Expansion of the a-CRISPRs shown in panel B at 7, 14 or 21 days post HHPV-2 infection (dpi). DNA from the infected (+) or uninfected (–) DF60PA cells was used as the positive or negative control. Three replicates were tested for each mutant, and each gel shows a representative result. The ∼300-bp (parental) and larger-sized (expanded) bands were relatively quantified for the 21-dpi (days post infection) samples, and the percentage of expanded PCR products in each lane is shown in the histogram. Lane Ms, dsDNA size markers.